Electroporation chamber including an electrode having a continuous, crystalline metal nitride coating

ABSTRACT

An improved electrode for use in generating an electrical field in a saline solution is provided. In particular, a continuous crystalline metal nitride coated electrode is provided for use in a variety of saline solution applications, such as in an electrophoresis device for separating proteins or nucleic acids or an electroporation apparatus for the encapsulation of biologically-active substances in various cell populations. A method and apparatus are provided for the encapsulation of biologically-active substances in red blood cells, characterized by an optionally automated, continuous-flow, self-contained electroporation system which allows withdrawal of blood from a patient, separation of red blood cells, encapsulation of a biologically-active substances in the cells, and optional recombination of blood plasma and the modified red blood cells thereby producing blood with modified biological characteristics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.09/618,654, filed Jul. 18, 2000, now U.S. Pat. No. 6,485,961, which is adivisional of U.S. patent application Ser. No. 08/760,515, filed Dec. 5,1996, now U.S. Pat. No. 6,090,617, issued Jul. 18, 2000.

TECHNICAL FIELD

The present invention relates to improved electrodes for use ingenerating an electrical field in a saline solution. In particular, theinvention relates to erosion resistant electrodes for use in a varietyof applications, such as in an electroporation device for theencapsulation of biologically-active substances in various cellpopulations. More particularly, the present invention relates toimproved electrodes for use in a method and apparatus for theencapsulation of allosteric effectors of hemoglobin in erythrocytes byelectroporation to achieve therapeutically desirable changes in thephysical characteristics of the intracellular hemoglobin.

BACKGROUND OF THE INVENTION

The present invention provides that an electrode surface may beprotected from wear, such as erosion and pitting, due to internallygenerated electrical signals occurring in a saline solution. Inparticular, a pulsed electrical signal such as generated by theelectroporation device described herein, normally causes acceleratederosion and inoperability of the electrodes, and furthermorecontaminates the solution and cells with metal ions. The presentinvention provides electrodes that can be subjected to frequent pulsesof electrical charge in a saline solution, as in an electroporationapparatus, and have substantially increased useful terms overconventional electrodes, without contamination of the products ofinterest.

Previous powdered porous metal nitride coatings, such as titaniumnitride (TiN), on electrodes used in gaseous environments have notaddressed the unique problems associated with electrodes used togenerate electric fields in an aqueous saline solution. In particular,the advantages previously taught for using an electrode having apowdered porous nitride coating are disadvantageous when used in aqueousbiological saline environments. The porosity of such prior art electrodecoatings does not serve to protect the exposed portions of the electrodesurface from surface ion erosion and pitting which are normallyaccelerated during electric signal emission in an aqueous salinesolution.

The use of a metal nitride coating has previously been taught to protectsurgical implants or instruments used in a biological system fromcorrosion and wearing due to externally generated forces, such as saltsor friction. However, when using electrodes in an aqueous biologicalsaline environment, internally delivered forces of charged particles(electrons and protons) emanating from the surface of the electrodecause accelerated pitting and erosion of the metal surface of theelectrode. Nitriding electrode surfaces has been proposed for improvingsignal detection in biological systems, such as in pacemaker detectionof intracardiac signals, however, not for electric signal generation orstimulation in biological systems, which presents the unique pitting anderosion problems described above. More specifically, the unique demandson a pair of electrodes sending rapid and reversing pulses of highvoltage electrical signals in an electroporation chamber, as describedherein, pose a problem heretofore unsolved in the art.

In the vascular system of an adult human being, blood has a volume ofabout 5 to 6 liters. Approximately one half of this volume is occupiedby cells, including red blood cells (erythrocytes), white blood cells(leukocytes), and blood platelets. Red blood cells comprise the majorityof the cellular components of blood. Plasma, the liquid portion ofblood, is approximately 90 percent water and 10 percent various solutes.These solutes include plasma proteins, organic metabolites and wasteproducts, and inorganic compounds.

The major function of red blood cells is to transport oxygen from thelungs to the tissues of the body, and transport carbon dioxide from thetissues to the lungs for removal. Very little oxygen is transported bythe blood plasma because oxygen is only sparingly soluble in aqueoussolutions. Most of the oxygen carried by the blood is transported by thehemoglobin of the erythrocytes. Erythrocytes in mammals do not containnuclei, mitochondria or any other intracellular organelles, and they donot use oxygen in their own metabolism. Red blood cells contain about 35percent by weight hemoglobin, which is responsible for binding andtransporting oxygen.

Hemoglobin is a protein having a molecular weight of approximately64,500 Daltons. It contains four polypeptide chains and four hemeprosthetic groups in which iron atoms are bound in the ferrous state.Normal globin, the protein portion of the hemoglobin molecule, consistsof two α chains and two β chains. Each of the four chains has acharacteristic tertiary structure in which the chain is folded. The fourpolypeptide chains fit together in an approximately tetrahedralarrangement, to constitute the characteristic quaternary structure ofhemoglobin. There is one heme group bound to each polypeptide chainwhich can reversibly bind one molecule of molecular oxygen. Whenhemoglobin combines with oxygen, oxyhemoglobin is formed. When oxygen isreleased, the oxyhemoglobin is reduced to deoxyhemoglobin.

Delivery of oxygen to tissues depends upon a number of factorsincluding, but not limited to, the volume of blood flow, the number ofred blood cells, the concentration of hemoglobin in the red blood cells,the oxygen affinity of the hemoglobin and, in certain species, on themolar ratio of intraerythrocytic hemoglobins with high and low oxygenaffinity. The oxygen affinity of hemoglobin depends on four factors aswell, namely: (1) the partial pressure of oxygen; (2) the pH; (3) theconcentration of the allosteric effective 2,3-diphosphoglycerate (DPG)in the hemoglobin; and (4) the concentration of carbon dioxide. In thelungs, at an oxygen partial pressure of 100 mm Hg, approximately 98% ofcirculating hemoglobin is saturated with oxygen. This represents thetotal oxygen transport capacity of the blood. When fully oxygenated, 100ml of whole mammalian blood can carry about 21 ml of gaseous oxygen.

The effect of the partial pressure of oxygen and the pH on the abilityof hemoglobin to bind oxygen is best illustrated by examination of theoxygen saturation curve of hemoglobin. An oxygen saturation curve plotsthe percentage of total oxygen-binding sites of a hemoglobin moleculethat are occupied by oxygen molecules when solutions of the hemoglobinmolecule are in equilibrium with different partial pressures of oxygenin the gas phase.

The oxygen saturation curve for hemoglobin is sigmoid. Thus, binding thefirst molecule of oxygen increases the affinity of the remaininghemoglobin for binding additional oxygen molecules. As the partialpressure of oxygen is increased, a plateau is approached at which eachof the hemoglobin molecules is saturated and contains the upper limit offour molecules of oxygen.

The reversible binding of oxygen by hemoglobin is accompanied by therelease of protons, according to the equation:

HHb⁺+O₂⇄HbO₂+H⁺

Thus, an increase in the pH will pull the equilibrium to the right andcause hemoglobin to bind more oxygen at a given partial pressure. Adecrease in the pH will decrease the amount of oxygen bound.

In the lungs, the partial pressure of oxygen in the air spaces isapproximately 90 to 100 mm Hg and the pH is also high relative to normalblood pH (up to 7.6). Therefore, hemoglobin will tend to become almostmaximally saturated with oxygen in the lungs. At that pressure and pH,hemoglobin is approximately 98 percent saturated with oxygen. On theother hand, in the capillaries in the interior of the peripheraltissues, the partial pressure of oxygen is only about 25 to 40 mm Hg andthe pH is also relatively low (about 7.2 to 7.3). Because muscle cellsuse oxygen at a high rate thereby lowering the local concentration ofoxygen, the release of some of the bound oxygen to the tissue isfavored. As the blood passes through the capillaries in the muscles,oxygen will be released from the nearly saturated hemoglobin in the redblood cells into the blood plasma and thence into the muscle cells.Hemoglobin will release about a third of its bound oxygen as it passesthrough the muscle capillaries, so that when it leaves the muscle, itwill be only about 64 percent saturated. In general, the hemoglobin inthe venous blood leaving the tissue cycles between about 65 and 97percent saturation with oxygen in its repeated circuits between thelungs and the peripheral tissues. Thus, oxygen partial pressure and pHfunction together to effect the release of oxygen by hemoglobin

A third important factor in regulating the degree of oxygenation ofhemoglobin is the allosteric effector 2,3-diphosphoglycerate (DPG). DPGis the normal physiological effector of hemoglobin in mammalianerythrocytes. DPG regulates the oxygen-binding affinity of hemoglobin inthe red blood cells in relationship to the oxygen partial pressure inthe lungs. In general, the higher the concentration of DPG in the cell,the lower the affinity of hemoglobin for oxygen.

When the delivery of oxygen to the tissues is chronically reduced, theconcentration of DPG in the erythrocytes is increased in normalindividuals. For example, at high altitudes the partial pressure ofoxygen is significantly less. Correspondingly, the partial pressure ofoxygen in the tissues is less. Within a few hours after a normal humansubject moves to a higher altitude, the DPG level in the red blood cellsincreases, causing more DPG to be bound and the oxygen affinity of thehemoglobin to decrease. Increases in the DPG level of red cells alsooccur in patients suffering from hypoxia. This adjustment allows thehemoglobin to release its bound oxygen more readily to the tissues tocompensate for the decreased oxygenation of hemoglobin in the lungs. Thereverse change occurs when people acclimated to high altitudes anddescend to lower altitudes.

As normally isolated from blood, hemoglobin contains a considerableamount of DPG. When hemoglobin is “stripped” of its DPG, it shows a muchhigher affinity for oxygen. When DPG is increased, the oxygen bindingaffinity of hemoglobin decreases. A physiologic allosteric effector suchas DPG is therefore essential for the normal release of oxygen fromhemoglobin in the tissues.

While DPG is the normal physiologic effector of hemoglobin in mammalianred blood cells, phosphorylated inositols are found to play a similarrole in the erythrocytes of some birds and reptiles. Although IHP isunable to pass through the mammalian erythrocyte membrane, it is capableof combining with hemoglobin of mammalian red blood cells at the bindingsite of DPG to modify the allosteric conformation of hemoglobin, theeffect of which is to reduce the affinity of hemoglobin for oxygen. Forexample, DPG can be replaced by inositol hexaphosphate (IHP), which iseven more potent than DPG in reducing the oxygen affinity of hemoglobin.IHP has a 1000-fold higher affinity to hemoglobin than DPG (R. E.Benesch et al., Biochemistry, Vol. 16, pages 2594-2597 (1977)) andincreases the P₅₀ of hemoglobin up to values of 96.4 mm Hg at pH 7.4,and 37 degrees C. (J. Biol. Chem., Vol. 250, pages 7093-7098 (1975)).

The oxygen release capacity of mammalian red blood cells can be enhancedby introducing certain allosteric effectors of hemoglobin intoerythrocytes, thereby decreasing the affinity of hemoglobin for oxygenand improving the oxygen economy of the blood. This phenomenon suggestsvarious medical applications for treating individuals who areexperiencing lowered oxygenation of their tissues due to the inadequatefunction of their lungs or circulatory system.

Because of the potential medical benefits to be achieved from the use ofthese modified erythrocytes, various techniques have been developed inthe prior art to enable the encapsulation of allosteric effectors ofhemoglobin in erythrocytes. Accordingly, numerous devices have beendesigned to assist or simplify the encapsulation procedure. Theencapsulation methods known in the art include osmotic pulse (swelling)and reconstitution of cells, controlled lysis and resealing,incorporation of liposomes, and electroporation. Current methods ofelectroporation make the procedure commercially impractical on a scalesuitable for commercial use.

The following references describe the incorporation of polyphosphatesinto red blood cells by the interaction of liposomes loaded with IHP:Gersonde, et al., “Modification of the Oxygen Affinity of IntracellularHaemoglobin by Incorporation of Polyphosphates into Intact Red BloodCells and Enhanced O2 Release in the Capillary System”, Biblthca.Haemat., No. 46, pp. 81-92 (1980); Gersonde, et al., “Enhancement of theO2 Release Capacity and of the Bohr-Effect of Human Red Blood Cellsafter Incorporation of Inositol Hexaphosphate by Fusion withEffector-Containing Lipid Vesicles”, Origins of Cooperative Binding ofHemoglobin, (1982); and Weiner, “Right Shifting of Hb-O₂ Dissociation inViable Red Cells by Liposomal Technique,” Biology of the Cell, Vol. 47,(1983).

Additionally, U.S. Pat. Nos. 4,192,869, 4,321,259, and 4,473,563 toNicolau et al. describe a method whereby fluid-charged lipid vesiclesare fused with erythrocyte membranes, depositing their contents into thered blood cells. In this manner, it is possible to transport allostericeffectors such as inositol hexaphosphate into erythrocytes, where, dueto its much higher binding constant IHP replaces DPG at its binding sitein hemoglobin.

In accordance with the liposome technique, IHP is dissolved in aphosphate buffer until the solution is saturated and a mixture of lipidvesicles is suspended in the solution. The suspension is then subjectedto ultrasonic treatment or an injection process, and then centrifuged.The upper suspension contains small lipid vesicles containing IHP, whichare then collected. Erythrocytes are added to the collected suspensionand incubated, during which time the lipid vesicles containing IHP fusewith the cell membranes of the erythrocytes, thereby depositing theircontents into the interior of the erythrocyte. The modified erythrocytesare then washed and added to plasma to complete the product.

The drawbacks associated with the liposomal technique include poorreproducibility of the IHP concentrations incorporated in the red bloodcells and significant hemolysis of the red blood cells followingtreatment. Additionally, commercialization is not practical because theprocedure is tedious and complicated.

In an attempt to solve the drawbacks associated with the liposomaltechnique, a method of lysing and the resealing red blood cells wasdeveloped. This method is described in the following publication:Nicolau, et al., “Incorporation of Allosteric Effectors of Hemoglobin inRed Blood Cells. Physiologic Effects,” Biblthca. Haemat., No. 51, pp.92-107, (1985). Related U.S. Pat. Nos. 4,752,586 and 4,652,449 to Roparset al. also describe a procedure of encapsulating substances havingbiological activity in human or animal erythrocytes by controlled lysisand resealing of the erythrocytes, which avoids the RBC-liposomeinteractions.

The technique is best characterized as a continuous flow dialysis systemwhich functions in a manner similar to the osmotic pulse technique.Specifically, the primary compartment of at least one dialysis elementis continuously supplied with an aqueous suspension of erythrocyteswhile the secondary compartment of the dialysis element contains anaqueous solution which is hypotonic with respect to the erythrocytesuspension. The hypotonic solution causes the erythrocytes to lyse. Theerythrocyte lysate is then contacted with the biologically activesubstance to be incorporated into the erythrocyte. To reseal themembranes of the erythrocytes, the osmotic and/or oncotic pressure ofthe erythrocyte lysate is increased and the suspension of resealederythrocytes is recovered.

In related U.S. Pat. Nos. 4,874,690 and 5,043,261 to Goodrich et al. arelated technique involving lyophilization and reconstitution ofred.blood cells is disclosed. As part of the process of reconstitutingthe red blood cells, the addition of various polyanions, includinginositol hexaphosphate, is described. Treatment of the red blood cellsaccording to the process disclosed results in a cell with unaffectedactivity. Presumably, the IHP is incorporated into the cell during thereconstitution process, thereby maintaining the activity of thehemoglobin.

In U.S. Pat. Nos. 4,478,824 and 4,931,276 to Franco et al. a secondrelated method and apparatus is described for introducing effectiveagents, including inositol hexaphosphate, into mammalian red blood cellsby effectively lysing and resealing the cells. The procedure isdescribed as the “osmotic pulse technique.” In practicing the osmoticpulse technique, a supply of packed red blood cells is suspended andincubated in a solution containing a compound which readily diffusesinto and out of the cells, the concentration of the compound beingsufficient to cause diffusion thereof into the cells so that thecontents of the cells become hypertonic. Next, a trans-membrane ionicgradient is created by diluting the solution containing the hypertoniccells with an essentially isotonic aqueous medium in the presence of atleast on desired agent to be introduced, thereby causing diffusion ofwater into the cells with a consequent swelling and an increases inpermeability of the outer membranes of the cells. This “osmotic pulse”causes the diffusion of water into the cells and a resultant swelling ofthe cells which increase the permeability of the outer cell membrane tothe desired agent. The increase in permeability of the membrane ismaintained for a period of time sufficient only to permit transport ofleast one agent into the cells and diffusion of the compound out of thecells.

Polyanions which may be used in practicing the osmotic pulse techniqueinclude pyrophosphate, tripolyphosphate, phosphorylated inositols,2,3-diphosphogly-cerate (DPG), adenosine triphosphate, heparin, andpolycarboxylic acids which are water-soluble, and non-disruptive to thelipid outer bilayer membranes of red blood cells.

The osmotic pulse technique has several shortcomings including low yieldof encapsulation, incomplete resealing, lose of cell content and acorresponding decrease in the life span of the cells. The technique istedious, complicated and unsuited to automation. For these reasons, theosmotic pulse technique has had little commercial success.

Another method for encapsulating various biologically-active substancesin erythrocytes is electroporation. Electroporation has been used forencapsulation of foreign molecules in different cell types including IHPred blood cells as described in Mouneimne, et a., “Stable rightwardshifts of the oxyhemoglobin dissociation curve induced by encapsulationof inositol hexaphosphate in red blood cells using electroporation,”FEBS, Vol. 275, No. 1, 2. pp. 117-120 (1990).

The process of electroporation involves the formation of pores in thecell membranes, or in any vesicles, by the application of electric fieldpulses across a liquid cell suspension containing the cells or vesicles.During the poration process, cells are suspended in a liquid media andthen subjected to an electric field pulse. The medium may beelectrolyte, non-electrolyte, or a mixture of electrolytes andnon-electrolytes. The strength of the electric field applied to thesuspension and the length of the pulse (the time that the electric fieldis applied to a cell suspension) varies according to the cell type. Tocreate a pore in a cell's outer membrane, the electric field must beapplied for such a length of time and at such a voltage as to create aset potential across the cell membrane for a period of time long enoughto create a pore.

Four phenomenon appear to play a role in the process of electroporation.The first is the phenomenon of dielectric breakdown. Dielectricbreakdown refers to the ability of a high electric field to create asmall pore or hole in a cell membrane. Once a pore is created, a cellcan be loaded with a biologically-active substances. The secondphenomenon is the dielectric bunching effect, which refers to the mutualself attraction produced by the placement of vesicles in a uniformelectric field. The third phenomenon is that of vesicle fusion. Vesiclefusion refers to the tendency of membranes of biological vesicles, whichhave had pores formed by dielectric breakdowns, to couple together attheir mutual dialectic breakdown sites when they are in close proximity.The fourth phenomenon is the tendency of cells to line up along one oftheir axis in the presence of high frequency electric fields. Thus,electroporation relates to the use in vesicle rotational prealignment,vesicle bunching and dielectric constant or vesicles for the purpose ofloading and unloading the cell vesicle.

Electroporation has been used effectively to incorporate allostericeffectors of hemoglobin in erythrocytes. In an article by Mouneimne, Yet al., “Stable Rightward Shifts of Oxyhemoglobin DisassociationConstant Induced by Encapsulation of Inositol Hexaphosphate in Red BloodCells Using Electroporation”, FEBS, Vol. 275, No. 1, 2, pages 11-120(November 1990). Mouneimne and his colleagues reported that right shiftsof the hemoglobin-oxygen dissociation in treated erythrocytes havingincorporated IHP can be achieved. Measurements at 24 and 48 hours afterloading with IHP showed a stable P₅₀ value indicating that resealing ofthe erythrocytes was permanent. Furthermore, it was shown that red bloodcells loaded with inositol hexaphosphate have a normal half life ofeleven days. However, the results obtained by Mouneimne and hiscolleagues indicate that approximately 20% of the retransfused cellswere lost within the first 24 hours of transfusion.

The electroporation methods disclosed in the prior art are not suitablefor processing large volumes of sample, nor use of a high or repetitiveelectric charge. Furthermore, the methods are not suitable for use in acontinuous or “flow” electroporation chamber. Available electroporationchambers are designed for static use only. Namely, processing of samplesby batch. Continuous use of a “static” chamber results in over heatingof the chamber and increased cell lysis. Furthermore, the existingtechnology is unable to incorporate a sufficient quantity of IHP in asufficient percentage of the cells being processed to dramaticallychange the oxygen carrying capacity of the blood. In addition, the priorart methods require elaborate equipment and are not suited for loadingred blood cells of a patient at the point of care. Thus, the procedureis time consuming and not suitable for use on a commercial scale.

What is needed is a simple, efficient and rapid method for encapsulatingbiologically-active substances in erythrocytes while preserving theintegrity and biologic function of the cells. The potential therapeuticapplications of biologically altered blood cells suggests the need forsimpler, and more effective and complete methods of encapsulation ofbiologically-active substances, including allosteric effectors ofhemoglobin in intact erythrocytes.

There are numerous clinical conditions that would benefit fromtreatments that would increase tissue delivery of oxygen bound tohemoglobin. For example, the leading cause of death in the United Statestoday is cardiovascular disease. The acute symptoms and pathology ofmany cardiovascular diseases, including congestive heart failure,myocardial infarction, stroke, intermittent claudication, and sicklecell anernia, result from an insufficient supply of oxygen in fluidsthat bathe the tissues. Likewise, the acute loss of blood followinghemorrhage, traumatic injury, or surgery results in decreased oxygensupply to vital organs. Without oxygen, tissues at sites distal to theheart, and even the heart itself, cannot produce enough energy tosustain their normal functions. The result of oxygen deprivation istissue death and organ failure.

Although the attention of the American public has long been focused onthe preventive measures required to alleviate heart disease, such asexercise, appropriate dietary habits, and moderation in alcoholconsumption, deaths continue to occur at an alarming rate. Since deathresults from oxygen deprivation, which in turn results in tissuedestruction and/or organ dysfunction. One approach to alleviate thelife-threatening consequences of cardiovascular disease is to increaseoxygenation of tissues during acute stress. The same approach is alsoappropriate for persons suffering from blood loss or chronic hypoxicdisorders, such as congestive heart failure.

Another condition which could benefit from an increase in the deliveryof oxygen to the tissues is anemia. A significant portion of hospitalpatients experience anemia or a low “crit” caused by an insufficientquantity of red blood cells or hemoglobin in their blood. This leads toinadequate oxygenation of their tissues and subsequent complications.Typically, a physician can temporarily correct this condition bytransfusing the patient with units of packed red blood cells.

Enhanced blood oxygenation may also reduce the number of heterologoustransfusions and allow use of autologous transfusions in more case. Thecurrent method for treatment of anemia or replacement of blood loss istransfusion of whole human blood. It is estimated that three to fourmillion patients receive transfusions in the U.S. each year for surgicalor medical needs. In situations where there is more time, it isadvantageous to completely avoid the use of donor or heterologous bloodand instead use autologous blood.

Often the amount of blood which can be drawn and stored prior to surgerylimits the use of autologous blood. Typically, a surgical patient doesnot have enough time to donate a sufficient quantity of blood prior tosurgery. A surgeon would like to have several units of blood available.As each unit requires a period of several weeks between donations andcan not be done less than two weeks prior to surgery, it is oftenimpossible to sequester an adequate supply of blood. By processingautologous blood with IHP, less blood is required and it becomespossible to completely avoid the transfusion of heterologdus blood.

As IHP-treated red cells transport 2-3 times as much oxygen as untreatedred cells, in many cases, a physician will need to transfuse fewer unitsof IHP-treaded red cells. This exposes the patient to less heterologousblood, decreases the extent of exposure to viral diseases from blooddonors and minimizes immune function disturbances secondary totransfusions. The ability to infuse more efficient red blood cells isalso advantageous when the patients blood volume is excessive. In othermore severe cases, where oxygen transport is failing, the ability torapidly improve a patient's tissue oxygenation is life saving.

Although it is evident that methods of enhancing oxygen delivery totissues have potential medical applications, currently there are nomethods clinically available for increasing tissue delivery of oxygenbound to hemoglobin. Transient, 6 to 12 hour elevations of oxygendeposition have been described in experimental animals using either DPGor molecules that are precursors of DPG. The natural regulation of DPGsynthesis in vivo and its relatively short biological half-life,however, limit the DPG concentration and the duration of increasedtissue PO₂, and thus limit its therapeutic usefulness.

What is needed is a simple, efficient and rapid method for encapsulatingbiologically-active substances, such as IHP, in erythrocytes withoutdamaging the erythrocytes.

SUMMARY OF THE INVENTION

The present invention provides improved electrodes for use in generatingan electrical field in a saline solution. In particular, the inventionrelates to continuous crystalline metal nitride coated electrodes foruse in a variety of saline solution applications. For example, theelectrodes of the invention can be used in an electrophoresis device forseparating particles such as proteins or nucleic acids. Additionally,the present invention relates to improved electrodes for use in anelectroporation apparatus for the encapsulation of biologically-activesubstances in various cell populations. Electrodes of the presentinvention have substantially longer useful lives than conventionalelectrodes, due to their increased resistance to erosion and pittingnormally caused by electrical signals emanating therefrom. Additionally,the products of devices employing such electrodes have substantiallyfewer metallic contaminates associated therewith.

More specifically, the present invention provides an electroporationchamber that may form part of an automated, self-contained, flowapparatus for encapsulating compounds or compositions, such as inositolhexaphosphate, in red blood cells, thereby reducing the affinity of thehemoglobin for oxygen and enhancing the delivery of oxygen by red bloodcells to tissues. Encapsulation is preferably-achieved byelectroporation; however, it is contemplated that other methods ofencapsulation may be used in practicing the present invention. Themethod and apparatus, including the electroporation chamber, of thepresent invention, is equally suited to the encapsulation of a varietyof biologically-active substances in various cell populations.

The apparatus and method of the present invention is suited to theincorporation of a variety of biologically-active substances in cellsand lipid vesicles. The method, apparatus and chamber of the presentinvention may be used for introducing a compound or biologically-activesubstance into a vesicle whether that vesicle is engineered or naturallyoccurring. For example, the apparatus, method, and chamber of thepresent invention may be used to introduce IHP into erythrocytes.

The encapsulation of inositol hexaphosphate in red blood cells byelectroporation according to the present invention results in asignificant decrease in the hemoglobin affinity for oxygen withoutaffecting the life span, ATP levels, K+ levels, or normal rheologicalcompetence of the cells. In addition, the Bohr effect is not alteredexcept to shift the O₂ binding curve to the right. Lowering the oxygenaffinity of the erythrocytes increases the capacity of erythrocytes todissociate the bound oxygen and thereby improves the oxygen supply tothe tissues. Enhancement of the oxygen-release capacity of erythrocytesbrings about significant physiological effects such as a reduction incardiac output, an increase in the arteriovenous differences, andimproved tissue oxygenation.

The modified erythrocytes prepared in accordance with the presentinvention, having improved oxygen release capacities, may find their usein situations such as those illustrated below:

1. Under conditions of low oxygen-partial pressure, such as at highaltitudes;

2. When the oxygen exchange surface of the lung is reduced, such asoccurs in emphysema;

3. When there is an increased resistance to oxygen diffusion in thelung, such as occurs in pneumonia or asthma;

4. When there is a decrease in the oxygen-transport capacity oferythrocytes, such as occurs with erythropenia or anemia, or when anarteriovenous shunt is used;

5. To treat blood circulation disturbances, such as arteriosclerosis,thromboembolic processes, organ infarct, congestive heart failure,cardiac insufficiency or ischemia;

6. To treat conditions of high, oxygen affinity of hemoglobin, such ashemoglobin mutations, chemical modifications of N-terminal amino acidsin the hemoglobin-chains, or enzyme defects in erythrocytes;

7. To accelerate detoxification processes by improving oxygen supply;

8. To decrease the oxygen affinity of conserved blood; or

9. To improve the efficacy of various cancer treatments.

According to the method and apparatus of the present invention, it ispossible to produce modified erythrocytes which contribute to animproved oxygen economy of the blood. These modified erythrocytes areobtained by incorporation of allosteric effectors, such as IHP, byelectroporation of the erythrocyte membranes.

The incorporation of the biologically-active substances into the cellsin accordance with the method of the present invention, including theencapsulation of allosteric effectors of hemoglobin into erythrocytes,is conducted extracorporally via an automated, flow electroporationapparatus. Briefly, a cell suspension is introduced into the separationand wash bowl chamber of the flow encapsulation apparatus. The cells areseparated from the suspension, washed and resuspended in a solution ofthe biologically-active substance to be introduced into the cell. Thissuspension is introduced into the electroporation chamber and thenincubated. Following electroporation and incubation, the cells arewashed and separated. A contamination check is optionally conducted toconfirm that all unencapsulated biologically-active substance has beenremoved. Then, the cells are prepared for storage or reintroduction intoa patient.

In accordance with the present invention and with reference to thepreferred embodiment, blood is drawn from a patient, the erythrocytesare separated from the drawn blood, the erythrocytes are modified by theincorporation of allosteric effectors and the modified erythrocytes andblood plasma is reconstituted. In this manner, it is possible to prepareand store blood containing IHP-modified erythrocytes.

The apparatus of the present invention provides an improved method forthe encapsulation of biologically-active substances in cells includingan apparatus which is self-contained and therefore sterile, an apparatuswhich can process large volumes of cells within a shortened time period,an apparatus having improved contamination detection, cooling andincubation elements, an apparatus is entirely automated and which doesnot require the active control of a technician once a sample isintroduced into the apparatus.

Thus, it is an object of the present invention to provide improvedelectrodes for generating an electrical signal in a saline solution.

It is an object of the present invention to provide improved electrodeswhich emit more reliable electrical field patterns and have longeruseful terms.

It is an object of the present invention to provide products made bydevices employing the improved electrodes which have minimalcontaminates associated therewith.

It is an object of the present invention to provide an automated,continuous flow or static volume encapsulation apparatus having theimproved electrodes.

It is a further object of the present invention to provide an automated,continuous flow electroporation apparatus having the improvedelectrodes.

It is a further object of the present invention to provide such acontinuous flow encapsulation apparatus or electroporation device whichproduces a homogenous population of loaded cells or vesicles.

It is another object of the present invention to provide a method andapparatus that allows continuous encapsulation of biologically-activesubstances in a population of cells or vesicles.

It is a further object of the present invention to provide a method andapparatus that achieves the above-defined objects, features, andadvantages in a single cycle.

It is another object of the present invention to provide an improved andmore efficient method of encapsulating biologically active substances incells than those methods currently available.

Other objects, features, and advantages of the present invention willbecome apparent upon reading the following detailed description of thepreferred embodiment of the invention when taken in conjunction with thedrawings and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a first embodiment of a continuous flowencapsulation apparatus.

FIG. 2 is a schematic diagram of a second embodiment of a continuousflow encapsulation apparatus.

FIG. 3 is a top view of a first embodiment of the flow electroporationchamber with electrodes.

FIG. 4 is a top view of a first embodiment of the flow electroporationchamber without electrodes.

FIG. 5 is a side view of a first embodiment of the flow electroporationchamber.

FIG. 6 is an end view of a first embodiment of the flow electroporationchamber.

FIG. 7 is a side view of an electrode for use with the first embodimentof the flow electroporation chamber.

FIG. 8 is a front view of the electrode of FIG. 7.

FIG. 9 is an exploded perspective view of a second embodiment of theflow electroporation chamber.

FIG. 10 is a perspective view of the flow electroporation chamber ofFIG. 9 with the chamber being assembled.

FIG. 11 is a graph comparing the effect of various field strengths,under static or flow conditions, on the % oxygenation ofIHP-encapsulated red blood cells.

FIG. 12 is a table comparing the effects of various field strengths,under static or flow conditions, on the P₅₀ value of IHP-encapsulatedred blood cells.

FIG. 13 is a table comparing the survival rates of red blood cellssubjected to electroporation under static and flow conditions at variousfield strengths.

FIG. 14 is a front elevation view of a support member of anelectroporation chamber according to a third embodiment of the presentinvention.

FIG. 15 is a cross-sectional view of the support member of FIG. 14 takenalong line 15—15 of FIG. 14.

FIG. 16 is an enlarged view of the section indicated by the circle 16 ofFIG. 15.

FIG. 17 is an exploded perspective view of the electroporation chamberaccording to the third embodiment and support column to which thechamber is mounted.

FIG. 18 is a perspective view showing the electroporation chamber ofFIG. 17 mounted to the support column.

FIG. 19 is a front elevation view of the electroporation chamberaccording to the third embodiment mounted to a support column.

FIG. 20 is a perspective cut-away view of the electroporation chamberand support column of FIG. 19.

FIG. 21 is a schematic view of a self-contained electroporationapparatus comprising the electroporation chamber of FIGS. 14-20.

FIG. 22 is a graph showing the resistance of several IHP solutions.

FIG. 23 is a schematic diagram of a third embodiment of a continuousflow encapsulation apparatus.

FIG. 24 is a cutaway view of a cell washing apparatus.

FIG. 25 is a side view of the cell plate showing the ridges defining thelabyrinth and the tubing showing the recirculation of the cellsuspension.

FIG. 26 is a cutaway view of a second embodiment of a cell washingapparatus.

FIG. 27 is a side cutaway view of the elastomeric chamber.

FIG. 28 is a graph showing representative electroporation voltage.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides that an electrode may be protected fromelectrical signal aberrations and wear, such as erosion and pitting, dueto internally emanating electrical signals when in a saline solution.The invention further provides that the saline solution, and anybiological particles of interest therein, may also be spared fromundesirable contamination resulting from such erosion. In particular, apulsed signal such as generated by the electroporation device describedherein, normally causes accelerated erosion, unpredictability in theelectric field and inoperability of the electrodes. The presentinvention provides that compared to conventional electrodes not providedwith such a coating, including electrodes coated with metal nitride byconventional techniques, electrodes which are continuous crystallinemetal nitride coated and subjected to frequent, high voltage pulses ofelectrical charge in a saline solution, as in an electroporationapparatus, are capable of emitting more consistent and predictablepatterns of electrical fields, have substantially increased usefulterms, and provide products containing substantially fewer metalliccontaminates, which is an important feature for use of electrodes withliving cells, tissues or organs.

The present invention provides that electrodes may be protected fromerosion and pitting which occur under normal use when emitting anelectronic signal therefrom in a saline solution by providing on atleast a portion of the surface thereof a substantially continuouscrystalline metal nitride coating. By “saline solution” is meant anybiologically or non-biologically occurring salts, such as sodium orpotassium for example, which have formed ions in an at least partiallyaqueous environment. Examples of metal nitride coatings include titaniumnitride (TiN), titanium aluminum nitride (TiAIN), chromium nitride(CrN), zirconium nitride (ZrN) and other nitrides of transition metalsof group IV of the periodic chart, and mixtures or alloys thereof,distinguished by high hardness, good electrical and thermalconductivity, high resistance to oxidation, and low coefficient offriction with respect to steel. By “crystalline” is meant that the metalnitride coating is deposited so as to form a lattice of metal nitridecrystals thereon. By “continuous” is meant that the coating does notcontain holes, or pores, in the crystalline metal nitride coating on theportion of the surface of the electrode intended to be coated. Thecontinuous crystalline metal nitride coating forms a barrier on theelectrode that is substantially impermeable to ions, such as metal ionsfrom the electrode, but is permeable to electrons of the electricalfield.

Several techniques well-known to those skilled in the art may be usedfor the deposit of a continuous crystalline metal nitride coating onto asubstrate, such as physical vapor deposition (PVD), nitrogen ionimplantation, and plasma ion nitriding. The invention provides that thecrystalline metal nitride coating may be from about 0.1 to 10 micronsthick, wherein the coating is continuous. Using a PVD process, thethickness of the coating typically ranges from about 1 to 5 microns.Metal nitride coatings typically provide a hardness of about 2,000 to3,000 HV as determined by Vickers hardness test at 50 gf load, and anadhesion of about 60 to 70 N critical normal force required to detachthe coating as measured by scratch testing. Such a metal nitride coatingis available for deposit on an electrode from Multi-Arc, Inc. (Rockaway,N.J.) as an ION BOND coating.

A discussion of the biocompatible properties of such metal nitridecoatings can be found for example in Therin et al., “A histomorphometriccomparison of the muscular tissue reaction to stainless steel, puretitanium and titanium alloy implant materials,” J. Materials Science:Materials in Medicine 2 (1991) pgs 1-8. Generally, the metal nitridecoatings TiN, TiAIN, CrN and ZrN have been found to be non-toxic,non-mutagenic, non-irritating, non-hemolytic and non-pyrogenic.

The invention provides that an electrode may be continuously crystallinemetal nitride coated on at least one surface from which the electronicsignal emanates in a dielectric system. Additionally, the inventionprovides that the entire electrode may have a continuous crystallinemetal nitride coating. Furthermore, either or both the cathode and anodemay have a continuous crystalline metal nitride coating on one or moresurfaces. The invention contemplates that electrodes used in any salinesolution may be improved by a continuous crystalline metal nitridecoating, such as employed in an electroporation apparatus or anelectrophoresis apparatus, for example.

Conventional electrodes are typically constructed of a metal alloy, suchas stainless steel, which emit ionic particles of the metal (e.g. Fe++),in addition to electrons, when a current is passed therethrough. Thisphenomenon is accelerated when the electrodes are in contact with asaline solution, and results in erosion and pitting of the electrodes.Erosion and pitting of the electrodes cause the electric field to becomeaberrant, and ultimately the electrodes become useless, in addition tocontaminating the solution. The continuous crystalline metal nitridecoating on the electrodes of the present invention readily permits theconduction of electrons therethrough to form a consistent andpredictable electrical field in the saline solution. However, thecontinuous crystalline metal nitride coating on the electrodes inhibitsthe migration of ions from the surface of the electrodes, whichotherwise causes pitting and erosion on the surface of the electrodes.This aspect of the invention can not be achieved if the nitride coatingis powdered, porous, or non-crystalline. Therefore, the inventionprovides that the presently disclosed electrodes, and the apparati inwhich they are used, have substantially increased predictability anduseful lifespans.

Furthermore, in the case of the below described electroporation device,the continuous crystalline metal nitride coating on the electrodesinhibits the inadvertent insertion of such metal ion particles into thetreated cells. In conventional electroporation device electrodes, theelectrical and saline induced erosion causes metal ionic particles toenter the solution, and ultimately into the porated cells. This aspectof the invention is extremely beneficial to reduce the potential forionic contamination of the final cell products. In the context of otherelectrode containing devices, such as an electrophoresis apparatus, thepresent invention also minimizes the contamination of metal ions in theproducts of interest, e.g. a polyacrylamide gel.

The present invention further provides an automated, self-contained,flow apparatus for encapsulating allosteric compounds or compositions,such as inositol hexaphosphate, in cells, such as red blood cells. Inone embodiment, the apparatus of the present invention combines thefeatures of a plasmaphoresis apparatus with those of a flowelectroporation apparatus to form an automated, self-contained flowelectroporation device. The present invention further comprises a newflow electroporation chamber that allows use of the chamber under flowrather than static conditions. It is contemplated that the method andapparatus, including the electroporation chamber of the presentinvention, may be used to encapsulate a variety of biologically-activesubstances in diverse cell populations using the improved, continuouscrystalline metal nitride coated electrodes of the present invention.The invention further contemplates that the continuous crystalline metalnitride coated electrodes may be used in any saline solution, includingbut not limited to the field of electrophoresis for the separation ofbiological particles, e.g. proteins or nucleic acids.

Additionally, the present invention provides a population of modifiedcells having physical characteristics that make the cells particularlyuseful for treating conditions which demand or benefit from an increasein the delivery of oxygen to the tissues. In accordance with the methodof the present invention, a homogenous population of IHP loaded redblood cells can be obtained with reduced contamination and a reducedpropensity to lyse following encapsulation. The treated red blood cellsexhibit normal life spans in circulation. Using the present invention,red blood cells of a patient in need of the treatment can be quicklyloaded and returned to the patient's circulation.

Related International Application No. PCT/US94/03189, filed Mar. 23,1994, which is a continuation-in-part of U.S. application Ser. No.035,467, filed Mar. 23, 1993, are hereby incorporated by reference.

The method of operation of the apparatus of the present invention isdescribed below with reference to the preferred use of the apparatus,i.e., the encapsulation of allosteric effectors of hemoglobin in redblood cells. Inositol hexaphosphate is the preferred allosteric effectorto be used with the present invention. Other sugar phosphates, such asinositol pentaphosphate, inositol tetraphosphate, inositol triphosphate,inositol diphosphate and diphosphatidyl inositol diphosphate, can alsobe used. Other suitable allosteric effectors include polyphosphates suchas nucleotide triphosphates, nucleotide diphosphates, nucleotidemonophosphates, and alcohol phosphate esters. In case of certainmutations of hemoglobin, e.g. “Zurich” hemoglobin, organic anions suchas polycarboxylic acids can be used as allosteric effectors. Finally, itis possible to use inorganic anions such as hexacyano ferrate, phosphateor chloride as allosteric effectors.

Red blood cells that have been loaded with inositol hexaphosphateaccording to the present invention can be used to treat a wide varietyof diseases and disease states. The IHP loaded red blood cells madeaccording to the present invention can be administered to a patientundergoing a heart attack thereby increasing the oxygen delivery to theischemic heart tissue and, at the same time, reducing the cardiacoutput. The IHP-loaded red blood cells made according to the presentinvention also can be used to treat any ischemic condition including,but not limited to, “bleeding” anemia, surgical complications, stroke,diabetes, sickle cell disease, burns, intermittent claudication,emphysema, hypothermia, peripheral vascular disease, congestive heartfailure, angina, transient ischemic disease, disseminated intravascularcoagulation, adult respiratory distress syndrome (ARDS) and cysticfibrosis. A detailed description of the medical applications ofcompositions prepared in accordance with the method of the presentinvention is also provided below.

Continuous Flow Encapsulation Apparatus

The method of operation of the apparatus of the present invention isdescribed below with reference to the preferred use of the apparatus,i.e., the encapsulation of allosteric effectors of hemoglobin in redblood cells by electroporation. It is to be understood that theapparatus may be adapted to accommodate other cell populations orvesicles, and other biologically active substances. Additionally, theapparatus maybe adapted to utilize methods of encapsulation other thanelectroporation.

Briefly, in accordance with the present invention, a sample of blood isintroduced into the continuous flow encapsulation apparatus. If redblood cells are being collected, the blood can either be drawn directlyfrom a patient or can be previously drawn blood. The blood is initiallyseparated into red blood cells, plasma and white blood cells, and wasteproducts. The waste products include the diluent and various bloodsolutes remaining in the supernatant after centrifugation. They arestored in a waste reservoir within the apparatus. The blood plasma andwhite blood cells are also retained in a reservoir within the systemwhile the red blood cells are admixed with the substance to beencapsulated. The suspension of red blood cells is then subjected toelectroporation. Following electroporation, the red blood cells areincubated under conditions which allow the cells to reseal. They arethen processed and washed to eliminate exogenous, non-encapsulatedbiologically-active substances. When the cells have been processed, thered blood cells containing the encapsulated substances can be optionallyreconstituted with the blood plasma and white blood cells. Thereconstituted blood may then be returned directly to the patient or canbe stored for later use. Although described as discrete steps, theprocess is essentially continuous.

A first embodiment of the present invention is described with referenceto FIG. 1, which schematically illustrates the structure of thecontinuous flow encapsulation apparatus of the present invention.

In accordance with the present invention, a volume of whole blood isadmitted into the electroporation system 5 at input 11. The blood samplemay optionally be drawn directly from a patient into the electroporationsystem 5, or the blood may be drawn at an earlier time and stored priorto introduction into the system 5. Valve 12 is opened to admit thesample into the system 5. Simultaneously, valve 25 is opened and pump 22is engaged to admit an anti-coagulant from the anti-coagulant reservoir27. A suitable anticoagulant is heparin, although other anticoagulantscan be used. The preferred anticoagulant is ACD. Valves 15 and 36 arealso opened and pump 40 is engaged. The admixture of anticoagulant andwhole blood passes through a filter 18 and a pressure evaluation system19 that monitors the flow through the apparatus, and is collected in ablood separation and wash bowl 44 which is activated when pump 40 isengaged. A sensor indicates when the blood separation and wash bowl 44has been filled with red blood cells. When it has been filled, the bloodsupply is stopped. The steps involving separation of the bloodcomponents can be accomplished by a plasmaphoresis apparatus, such asthe plasmaphoresis apparatus manufactured by Haemonetics Corporation(Haemonetics Corporation, Braintree, Mass.).

As explained above, when pump 40 is engaged in a clockwise direction,the blood separation and wash bowl 44 is engaged and the anti-coagulantand whole blood suspension is centrifuged to separate the plasma, whiteblood cells, red blood cells, and waste. Valve 87 is opened to admit theplasma and while blood cells into the plasma reservoir 89.

Optionally, and dependent on the cell population being processed by theapparatus, the cells retained in the blood separation and wash bowl 44are then washed. Valves 33, 15, and 36 are opened to admit saline bufferfrom the diluent reservoir 30 into the blood separation and wash bowl 44which contains the red blood cells. Pump 40 is still engaged. The redblood cells are then washed and centrifuged. The preferred saline bufferis a 0.9% sodium chloride solution, although other physiologicallyisotonic buffers can be used to dilute and wash the red blood cells.Valve 54 is opened to admit the waste into the waste reservoir 57 duringthe washing process. Again, the waste is stored in the waste reservoir57 and the red blood cells are retained in the blood separation and washbowl 44. The wash process is repeated if necessary.

It has been found through experiments conducted with a variety ofchanges in pulse lengths and field strengths that square pulses resultin less-efficient encapsulation of IHP into human erythrocytes. Thecreation of large pores in the cell membrane appears to be insufficientfor the entry of extracellular IHP into red blood cells. This suggests amore complex process than the diffusion of IHP into the cells after thecreation of the pores. It is proposed that the electrical pulse has toaccomplish two tasks. The first is the generation of pores in the cellmembrane and the second is the active electrophoretic movement of theIHP through those pores into the red blood cell. This can beaccomplished through the use of high voltage square pulses (2.13 kV/cm,2 ms) immediately followed by a lower voltage exponential pulse (1.5 to1.75 kV/cm, 5 ms), which leads to an.increased encapsulation of IHP intored blood cells of up to 50% of the usual exponential pulse protocolencapsulation. The exponential pulse itself is well below theelectroporation threshold. Both tasks, namely pore formation andelectrophoretic movement, can be most effectively accomplished with useof exponential pulses. Another embodiment is to first expose the cellsto a high voltage square pulse and then a series of lower voltage pulseswhich tend to drive the IHP into the red blood cells resulting in a moreefficient loading of the IHP into the cells. In use, the cells travelingthrough the electroporation chamber of the present invention is exposedto a series of pulse trains. The pulse train is between 80 and 512pulses with the preferable number of pulses of 312 pulses. The polarityis then changed and a second pulse train is then applied to the cells.When the third set of pulses is applied, the polarity is again changed.For any given cell as it travels the length of the electroporationchamber, three to five pulse trains are applied reversing the polaritybetween each pulse train.

Following separation of the red blood cells, pump 40 is reversed, pump22 is turned off, valves 12, 15, 33, 36, 25, 87, and 54 are closed, andvalves 97 and 64 are opened. The IHP solution is pumped out of the IHPreservoir SO while, simultaneously, red blood cells are pumped out ofthe blood separation and wash bowl 44 towards the cooling coil 68. Thered blood cells and IMP solution are admixed in the tubing of theapparatus at junction 67 and then pumped through the cooling coil 68. Ina preferred embodiment of the present invention, and as explained indetail below, the IHP solution and red blood cells may be admixed in theseparation and wash bowl 44 before being admitted into the cooling coil68.

The preferred concentration of IHP in the solution is betweenapproximately 10 mMol and 100 mMol with a more preferred concentrationof approximately 22.5 to 50 mMol, and a most preferred concentration of35 mMol. The preferred concentration of KCl in the IHP solution isbetween approximately 10 mM and 5 mM. The preferred concentration ofMgCl₂is between approximately 2 mM and 0.5 mM. The preferredconcentration of sucrose in the IHP solution is between approximately67.5 mM and 270 mM. It is to be understood that other sugars or polymerscan be used as a substitute for sucrose.

The solutions that are used in the present invention are resistanceenhancing fluids. It is important to note that the IHP solution shouldhave a high resistivity and should have a minimum of electrolytes. TheIHP from Aldrich Chemical Company or from Matrea Chemical Company doesnot contain any sodium chloride and a minimum of other electrolytes andtherefore does not significantly decrease the resistivity of thesolution. The milliosmolarity of the solution should be betweenapproximately 300 and 500. The resistivity should be betweenapproximately 87 Ω·cm and 185 Ω·cm. The conductivity should be betweenapproximately 4 to 8 nS/cm. The practical salinity should be betweenapproximately 4 and 9 ppt and the NaCl equivalent should be betweenapproximately 4.5 and 9.0 ppt.

The hematocrit of the suspension is preferably between approximately 30and 80 with the most preferred hematocrit of approximately 40. It hasbeen determined from red cell responses that the high voltage should notexceed 800 volts in the static cell (whose gap is 0.4 cm), whichcorresponds to 2 kV/cm. For the flow cell, which has a 0.3 cm gap, thevoltage across the cell will be limited to 600 volts, (+/−300 v). Anumber of different electroporation fluid compositions have been tested.Table A lists six samples and their characteristics. The solution underE is the preferred electroporation solution. Pump 40 is designed to pumpboth red blood cells and IHP solution and can be adjusted so that thefinal hematocrit entering the cooling coil 68 can be predetermined.

TABLE A A^(a) B^(b) C^(c) D^(d) E^(e) CBR^(f) Conductivity 2.78 8.9211.2 8.67 7.07 16.8 (mS/cm) Resistivity 361 112 89.1 115 134 59.3(ohm-cm) mOsm 379 472 408 397 314 452 Practical 1.54 5.43 6.91 5.24 4.4511 Salinity (ppt) NaCl 1.71 5.43 6.76 5.25 4.59 10.2 Equivalent (ppt) pH7.39 7.346 7.185 7.316 7.4 7.42 Phytic Acid Aldrich Sigma AldrichAldrich Matreya Sigma (IHP) IHP IHP IHP IHP IHP IHP ^(a)10 mmol KCl, 2mm MgCl₂ 270 mmol Sucrose, 35 mmol IHP ^(b)Same as A except withpotassium salt of IHP ^(c)Iscove's Mod. Dulbecco's 125 mmol Su^(d)Dulbecco's phosphate buffered saline, 125 mmol Sucrose ^(e)5 mmolKCl, 1 mmol MgCl₂, 135 mmol sucrose ^(f)33 mmol K₂HPO₄, 7.0 mmolNaH₂PO₄, 30.6 mmol KCl, 6.4 mmol NaCl, 7.3 mmol sucrose, 5.0 mmol ATP

After mixing, the red blood cell-IHP suspension is then pumped through acooling coil 68. Cooling can be achieved with a water bath or with athermo-electric based cooling system. For example, cooling coil 68 isimmersed in a cooling bath in the cooling reservoir 69. When the redblood cell-IHP suspension passes through the cooling coil 68, thesuspension is cooled to a temperature of between approximately 1° C. and12° C., preferably approximately 4° C.

Cooling the red blood cells ensures the survival of the pore created inthe cell membrane during the electroporation process. The use of acooling coil aids in the speed of cooling by increasing the surface areaof the sample in contact with the cooling element. Optionally, thecooling coil can be surrounded by a thermo-electric heat pump.

Certain applications may require heating of the cell suspension prior toelectroporation. In such a case, a heating coil may replace the coolingcoil 68. The maximum temperature tolerated by red blood cells isapproximately 37° C.

A thermoelectric heat pump works by extracting thermal energy from aparticular region, thereby reducing its temperature, and then rejectingthe thermal energy into a “heat sink” region of higher temperature. Atthe cold junction, energy is absorbed by electrons as they pass from alow energy level in the p-type semiconductor element, to a higher energylevel in the n-type semiconductor element. The power supply provides theenergy to move the electrons through the system. At the hot junction,energy is expelled into a heat sink as electrons move from a high energylevel element (n-type) to a lower energy level element (p-type).

Thermoelectric elements are totally solid state and do not have movingmechanical parts or require a working fluid, as do vapor-cycle devices.However, thermoelectric heat pumps perform the same cooling functions asfreon-based vapor compression or absorption refrigerators.Thermoelectric heat pumps are highly reliable, small in size andcapacity, low cost, low weight, intrinsically safer than many othercooling devices, and are capable of precise temperature control.

The preferred thermoelectric heat pumps for use in the present inventionare manufactured by MELCOR Materials Electronic Products Corp. ofTrenton, N.J. The thermocouples are made of high performance crystallinesemiconductor material. The semiconductor material is bismuth telluride,a quaternary alloy of bismuth, tellurium, selenium, and antimony, dopedand processed to yield oriented polycrystalline semiconductors withproperties. The couples, connected in series electrically and inparallel thermally, are integrated into modules. The modules arepackaged between metallized ceramic plates to afford optimum electricalinsulation and thermal conduction with high mechanical strength incompression. Modules can be mounted in parallel to increase the heattransfer effect or can be stacked in multiple-stage cascades to achievehigh differential temperatures. Passing a current through the heat pumpgenerates a temperature differential across the thermocouples, withmaximum ratings of 70° C. and higher.

After cooling, the red blood cell-IHP suspension enters theelectroporation chamber 72 where an electric pulse is administered froma pulse generator 75 to the red blood cell-IHP suspension, causingopenings to form within the cell membranes of the red blood cells.Optionally, an automatic detection system will turn the pulse generator75 on when the chamber 72 is filled with red blood cell-IHP suspension.An electrical pulse is applied to the suspension every time the chamber72 is filled with unencapsulated cells. A conventional electroporationchamber may be used when the operation of the apparatus is static,namely, when single discrete batches of cells are processed. In apreferred embodiment of the present invention a flow electroporationchamber is used. In one embodiment, a flow electroporation chamber 72 isconstructed of clear polyvinyl chloride, and contains two opposingelectrodes spaced a distance of 7 mm apart. The distance between theelectrodes will vary depending on the flow volume and field strength.Preferably, the flow electroporation chamber 72 is disposable. Theelectroporation chamber may also be constructed of polysolfone, which ispreferably for use with certain sterilization procedures, such asautoclaving. A detailed description of the structure and construction ofthe flow electroporation chamber is provided below.

The red blood cell-IHP suspension passes between the two electrodes ofthe electroporation chamber 72. When a suspension of non-treated cellsenter the chamber 72, an electrical field of 1 to 3 KV/cm is created andmaintained for a period of 1 to 4 milliseconds, preferably for a periodof 2 milliseconds with a 1.8 ml flow chamber. Preferably, the IHP-redblood cell suspension is subjected to three high voltage pulses pervolume at a fieldstrength of approximately 2600 to 3200 V/cm per pulse.The pulse of current across the cell membranes causes an electricalbreakdown of the cell membranes, which creates pores in the membranes.IHP then diffuses into the cell through these pores.

Following electroporation, the red blood cell-IHP suspension enters anincubation chamber 78 where the suspension is incubated at roomtemperature for an incubation time of between approximately 15 minutesand 120 minutes with the preferred incubation time of 30 to 60 minutes.Optionally, the red blood cell-IHP suspension is incubated forapproximately 5 minutes at a temperature of approximately 37° C., and atleast 15 minutes at room temperature. The incubation chamber 78 mayoptionally be surrounded by a heating means 80. For example, the heatingmeans 80 can be a water bath or can be a thermoelectric heat pump.

Optionally, the incubator 78 contains a resealing buffer which aids inresealing and reconstitution of the red blood cells. The preferredcomposition of the resealing buffer is provided below in Table B:

TABLE B RESEALING BUFFER I. Combine Sodium chloride 150 mMol Potassiumchloride  8 mMol Sodium phosphate  6 mMol Magnesium sulfate  2 mMolGtucose  10 mMol Adenine  1 mMol Inosine  1 mMol Penicillin G 500units/ml Chloramphenicol 0.1 mg/ml II. Add BSA 3.5% Calcium  2 mMolchloride

In the preferred embodiment of the present invention, no resealingbuffer is used.

Following incubation, valve 51 is opened and pump 40 is engaged and thered blood cell-IHP suspension is returned to the blood separation andwash bowl 44 from the incubation chamber 78. The excess IHP solution isremoved from the red blood cell suspension by centrifugation. The wasteIHP solution is directed to waste reservoir 57. Valves 33, 15 and 36 arethen opened to admit a volume of diluent into the blood separation andwash bowl 44. A diluent that can be used in the present invention isshown in Table C.

TABLE C DILUENT BUFFER I. Combine Sodium chloride 0.9% Magnesiumchloride  2 mM Calcium chloride  2 mM Magnesium sulfate  2 mMol Glucose10 mMol 0.1% Penicillin (Optional) 0.1% 0.1% Streptomycin (Optional)0.1%

The red blood cell-IHP suspension is then centrifuged and thesupernatant is discarded in the waste reservoir 57 through valve 54leaving the red blood cells in the blood separation and wash bowl 44. Asaline buffer is added to the modified red blood cells from the diluentreservoir 30. The cells are washed and the supernatant is discardedfollowing centrifugation. The wash process is repeated if needed.

Optionally, as the waste is removed from the separation and wash bowl 44it passes through a contamination detector 46 to detect any free IHP inthe waste solution thereby confirming that exogenous non-encapsulatedIHP has been removed from the modified red blood cells. Thecontamination detection system relies on optical changes in the washingbuffer. After the modified red blood cells have been washed andcentrifuged, the supernatant passes through the contamination detector64 before it is deposited in the waste reservoir 57. If exogenous,non-encapsulated IHP remains in the washing buffer, The discardedsolution will be turbid. The turbidity is due to the reaction of IHPwith calcium, which is a component of the wash buffer. The contaminationdetector 46 uses an optical detection system. Preferably, the lightsource is an LED and the detector is a photodiode. The voltagedifference of the photodiode will indicate the amount of IHP in the washsolution. The contamination detector 46 is optional.

Following washing, the IHP-red blood cell product is optionallyreconstituted with the plasma and white blood cells which had beenretained in reservoir 89. The treated red blood cells may be collectedin a reinjection bag, either in a preservation media or in theautologous plasma of the patient.

The IHP-loaded red blood cells obtained can be administered directlyback into the patient or the cells can be stored for later use. The IHPin the red blood cells is not released during the normal storage time.

A preferred embodiment of the present invention is described withreference to FIG. 2, which schematically illustrates the structure ofthe continuous flow encapsulation apparatus of the present invention.Again, the method of operation of the apparatus is described withreference to the preferred use of the apparatus, i.e., the encapsulationof allosteric effectors of hemoglobin in red blood cells byelectroporation. It is to be understood that the apparatus may beadapted to accommodate other cell populations or vesicles, and otherbiologically active substances. Additionally, the apparatus maybeadapted to include other methods of encapsulation.

In accordance with the present invention, a sample of whole blood isadmitted into the electroporation system 10 at input 11. Valve 12 isopened to admit the sample into the system 10. Simultaneously, valve 25is opened and pump 22 is engaged to admit an anti-coagulant from theanti-coagulant reservoir 27. Valves 15 and 36 are also opened and pump40 is engaged.

The admixture of anticoagulant and whole blood passes through a filter18 and a pressure evaluation system 19, and is collected in a bloodseparation and wash bowl 44 which is activated when pump 40 is engaged.A sensor indicates when the blood separation and wash bowl 44 has beenfilled with red blood cells.

When pump 40 is engaged in a clockwise direction, the blood separationand wash bowl 44 is engaged and the anti-coagulant and whole bloodsuspension is centrifuged to separate the plasma, white blood cells, redblood cells, and waste. Valve 87 is opened to admit the plasma and whiteblood cells into the plasma reservoir 89.

Optionally, the cells retained in the separation and wash bowl 44 arethen washed and centrifuged. Valves 33, 35, 15, and 36 are opened toadmit saline buffer from the diluent reservoir 30 into the bloodseparation and wash bowl 44 which contains the red blood cells. Valve 12is closed and pump 40 remains engaged.

During washing, valve 54 is opened to admit the waste into the wastereservoir 57 during the washing process. Again, the waste is stored inthe waste reservoir 57 and the red blood cells are retained in the bloodseparation and wash bowl 44. The wash process is repeated if necessary.A contamination detection system may optionally be installed between theseparation and wash bowl 44 and the waste reservoir 57 to control thewash process.

Following separation of the red blood cells, pump 40 is reversed, pump22 is turned off, valves 12, 15, 33, 35, 36, 25, 87, and 54 are closed,and valve 97 is opened. If the cells were washed, pump 22 was previouslyturned off and valves 12 and 25 had been closed. The IHP solution ispumped out of the IHP reservoir 50 and into the separation and wash bowl44 containing the red blood cells. There, the red blood cells and IHPare admixed to form a suspension.

The preferred concentration of IHP in the solution is betweenapproximately 10 mMol and 100 mMol with a more preferred concentrationof approximately 23 to 35 mMol, and with a most preferred concentrationof 35 mMol. The preferred IHP solution comprises the followingcompounds, in the following concentrations:

35 mM IHP (Hexasodium salt) neutralized (Matreya Chemical Company)

5 mM KCl

1.0 mM MgCl₂

135 mM sucrose

The IHP from Aldrich Chemical Company does not contain any sodiumchloride and a minimum of other electrolytes and therefore does notsignificantly decrease the resistivity of the solution. It is to beunderstood that other solutions with high impedance can be used in thepresent invention and that the components of the solution are notcritical. As long as the osmotic properties of the solution are suchthat the cells, such as red blood cells are not damaged, and theresistivity of the solution is high, it is suitable for use in thepresent invention. Several compositions were tested for resistivity andare shown in FIG. 22. The “CBR Fluid” is shown in Table A.

The hematocrit of the suspension is preferably between approximately 30and 60 with the most preferred hematocrit of approximately 40. Pump 40is designed to pump both red blood cells and IHP solution and can beadjusted so that the final hematocrit entering the cooling coil 68 canbe predetermined.

After combining the red blood cells with the IHP solution, pump 40 isagain reversed, valve 97 is closed and valve 64 is opened. The red bloodcell-IHP suspension is then pumped through a thermoelectric cooling coil68. A blood bag from a blood warming set, such as the blood bag providedin the Fenwal® Blood Warming Set manufactured by Baxter HealthcareCorporation can be used as the cooling coil 68. When the red bloodcell-IHP suspension passes through the cooling coil 68 in the coolingreservoir 69, the suspension is cooled to a temperature of betweenapproximately 1° C. and 12° C., preferably approximately 4° C.Optionally, a pump may be added to the apparatus between the coolingcoil 68 and cooling reservoir 69, and the electroporation chamber 72, toensure a constant flow rate and compensate for fluctuation in volumethat occurs when the cooling coil 68 is filled.

Optionally, the pre-cooling step may be eliminated and the red bloodcell-IHP suspension may be directed to the electroporation chamber 72immediately after admixing. In such an instance, the cooling coil 68 andcooling reservoir 69 would be eliminated from the continuous flowencapsulation apparatus 10. Cooling prior to electroporation may not berequired if the temperature of the electroporation chamber issufficiently cool to maintain the cells suspension at 4° C.

After cooling, the red blood cell-IHP suspension enters theelectroporation chamber 72. The chamber 72 is maintained at atemperature of approximately 4° C. As the red blood cell-IHP suspensionpasses through the flow electroporation chamber 72, an electric pulse isadministered from a pulse generator 75 to the suspension causingopenings to form within the cell membranes of the red blood cells.

The red blood cell-IHP suspension passes between two electrodes of theelectroporation chamber 72. The electrodes of the present invention arepreferably coated on at least the surface from which the electric fieldemanates with a continuous crystalline metal nitride, as described inmore detail above. FIGS. 3 to 10 describe the electroporation chamber.In a preferred embodiment of the present invention, when a suspension ofnon-treated cells enters the chamber 72, the IHP-red blood cellsuspension is subjected to approximately three high voltage pulses pervolume or pulse trains per volume at a fieldstrength of approximately2600 to 3200 V/cm per pulse. It has been determined that forintroduction of IHP into blood, instead of a single pulse, a train ofshort pulses is more efficient in transporting IHP into the red bloodcell. The optimal number of pulses is between approximately 10 pulses to512 pulses per train with the preferable number being approximately 312pulses. It is also advantageous to change the polarity of the fieldbetween pulses or pulse trains. In FIG. 28, a representative two pulsetrain is shown. The charge created across the cell membranes causes abreakdown of the cell membrane, which creates pores in the membrane. IHPthen diffuses into the cell through these pores. In addition, althoughnot wanting to be bound to the following hypothesis, it is believed thatthe IHP is actually forced into the cell in the electric field.

During electroporation, an electrical field of 1 to 3 KV/cm is createdand maintained for a period of 1 to 4 milliseconds. The preferred pulselength is 3 to 4 milliseconds, with a most preferred pulse length of 2milliseconds. Pulse length or pulse train length is defined as l/e. At aflow rate of approximately 10.6 ml/minute, the preferred number ofpulses is 3 to 5, at the preferred pulse rate of 0.29 Hz. Thefieldstrength is defined as the voltage over the distance between theelectrodes. The distance between electrodes is measured in centimeters.The preferred electrical parameters are as follows:

pulse length or pulse train length=1.5 to 2.5 ms

fieldstrength=2.7 to 3 KV/cm

The electroporation chamber can optionally be a sensor in the sense thatthe resistivity of the cell solution that is traveling through theelectroporation chamber is monitored as the resistivity of the cellsolution changes, there is a feedback circuit that will adjust thepulsing of the cells to maintain optimum electroporation efficiency. Forexample, when electroporating blood in an IHP solution, differentsamples of blood may have different resistivity. By monitoring theresistivity of the blood, optimal pulse strengths and pulse timing canapplied based on the resistivity measurement. In addition, if a bubbleshould be introduced into the electroporation chamber, the feedbackcircuit will sense the presence of the bubble because of the change inresistivity, and will turn off the pulsing until the bubble exits thechamber.

Following electroporation, the red blood cell-IHP suspension enters anincubation chamber 78 where the suspension is incubated at roomtemperature for an incubation time of between approximately 10 minutesand 120 minutes with a preferred incubation time of 30 minutes.Optionally, the red blood cell-IHP suspension is incubated forapproximately 5 minutes at a temperature of approximately 37° C., and atleast 15 minutes at room temperature. The incubation chamber 78 may besurrounded by a heating means 80. Any heating means 80 can be used inpracticing the present invention. The preferred heating means 80 are awater bath or a thermoelectric heat pump.

Optionally, the incubator 78 contains a resealing buffer which aids inresealing and reconstitution of the red blood cells. In the preferredembodiment of the present invention, no resealing buffer is used.

Following incubation, the red blood cell-IHP suspension is returned tothe blood separation and wash bowl 44 when valve 51 is opened and pump40 is engaged. The excess IHP solution is removed from the red bloodcell suspension by centrifugation. The waste IHP solution is directed towaste reservoir 57. Valves 33, 37, 15 and 36 are then opened to admit avolume of post wash solution from reservoir 31 into the blood separationand wash bowl 44. In a preferred embodiment of the present invention,the post wash solution comprises a 0.9% NaCl solution, including 2.0 mMCaCl₂ and 2.0 mM MgCl₂. Any physiological saline may be used.

After addition of the post wash solution, the red blood cell-IHPsuspension is then centrifuged and the supernatant is discarded in thewaste reservoir 57 through valve 54 leaving the red blood cells in theblood separation and wash bowl 44. The wash process is repeated untilall unencapsulated IHP has been removed.

Optionally, as the waste is removed from the separation and wash bowl 44it passes through a contamination detector 46 to detect any free IHP inthe waste solution thereby confirming that exogenous non-encapsulatedIHP has been removed from the modified red blood cells. Thecontamination detector 46 is optional.

Following washing, the red blood cells containing IHP may bereconstituted with the plasma and white blood cells retained inreservoir 89. Pump 40 is engaged and valves 87, 36, and 92 are opened.The modified red blood cells and plasma and white blood cells are pumpedto reservoir 96. A filter may be installed between reservoir 96 andvalve 92 to remove any aggregates or other impurities from thereconstituted modified blood.

The IHP-loaded red blood cells obtained in accordance with the method ofthe present invention can be administered directly back into the patientor the cells can be stored for later use. The IHP in the red blood cellsis not released during the normal storage time.

It is contemplated that continuous flow encapsulation apparatus of thepresent invention may be modified to utilize other encapsulationmethods.

Furthermore, it is contemplated that the.continuous flow encapsulationapparatus may be adapted to process various diverse cell populations.Furthermore, the apparatus may be used to encapsulate biologicallyactive substances in artificial vesicles.

It is also contemplated that the continuous flow encapsulation apparatusof the present invention may be used to encapsulate a broad range ofbiologically active substances.

Flow Electroporation Chamber

During electroporation, the insertion rate of IHP is linearly dependenton the voltage administered to the cells. Generally, the higher thevoltage, the more IHP is encapsulated; however, cell lysis is alsoincreased and cell survival is decreased. The efficiency of anelectroporation system may be judged by cell survival afterelectroporation. Poor cell survival indicates very low efficiency. Theamplitude and duration of the electrical pulse is responsible for theelectric breakdown of the cell membrane and creates pores in the polecaps parallel to the electric field. Thus, the factors to be consideredin designing an electroporation system include the field strength, thepulse length and the number of pulses.

A perfect electroporation target is shaped like a sphere, so itsorientation does not effect the efficiency of the applied field. Whenthe target is spherical, a single pulse with a fieldstrength above thethreshold can electroplate 100% of the target. Red blood cells are diskshaped. Because of their shape and orientation in the electroporationchamber, only approximately 40% of the cells are electroplated during asingle pulse. To also electroporate the other 60%, the fieldstrength canbe increased. This increases the stress on the red blood cells in properorientation to the electric field and leads to lower survival rates ofthe cells.

To achieve more efficient encapsulation while reducing the incidence ofcell lysis and death, a flow-electroporation chamber utilizing shortduration multiple pulses was developed. With the flow-through ratesteady and a steady field voltage, it was determined that plurality ofpulses would insert maximal quantities of IHP with minimal 2 to 24 hourall lysis. A multiple-pulse system allows an increase in the cellsurvival rate without increasing the field strength. When amultiple-pulse system is used, orientation of the cells is not ascritical as it is when a system is a single pulse system is used. Thelower fieldstrength is much more gentle to the red blood cells. It ismuch easier to electroporate every single cell in the multiple pulsesystem, because the timing between the flow rate of the red blood cellsthrough the chamber and the electroporation pulses, and the orientationof the cells is not as crucial as in a single pulse system. The flowmultiple-pulse electroporation system also increases both the short termand the long term survival of red blood cells when compared to thesingle pulse method.

FIGS. 11 to 13 illustrate the effects of various field strengths, understatic or flow conditions, on the % oxygenation of IHP-encapsulated redblood cells over a range oxygen pressures: on the P₅₀ value ofIHP-encapsulated red blood cells (two concentrations of IHP solutionswere compared); and, on the survival rates of red blood cells subjectedto electroporation. All readings were taken 24 hours afterelectroporation. The results indicated that multiple pulses atcomparatively low fieldstrengths produce optimal encapsulation results.

A cooled electroporation chamber is preferred to keep the red bloodcells at a constant temperature during the electroporation process,thereby enhancing their survival rates. This is accomplished by removingthe excess heat created by the electrical pulse during theelectroporation process. The excess heat may be removed either bycooling the electrodes or cooling the entire flow electroporationchamber. In accordance with one embodiment of the present invention, theelectrodes themselves are cooled.

During the electroporation process, blood is pumped through an inlet inthe electroporation chamber and the red blood cells are subject to aseries of electrical pulses as they travel through the chamber. Theyexit out the other end of the chamber. The chamber can be made of anytype of insulating material, including, but not limited to, ceramic,Teflon, Plexiglas, glass, plastic, silicon, rubber or other syntheticmaterials. Preferably, the chamber is comprised of glass or polysulfone.Whatever the composition of the chamber, the internal surface of thechamber should be smooth to reduce turbulence in the fluid passingthrough it. The housing of the chamber should be non-conductive andbiologically inert. In commercial use, it is anticipated that thechamber will be disposable.

In one preferred embodiment of the present invention, the electrodesthat comprise part of the electroporation apparatus can be constructedfrom any type of electrically or thermally conductive hollow stockmaterial, including, but not limited to, brass, stainless steel, goldplated stainless steel, gold plated glass, gold plated plastic, or metalcontaining plastic. The surface of the electrode can be gold plated.Gold plating serves to eliminate oxidation and reduces the collection ofhemoglobin and other cell particles at the electrodes. The surface ofthe electrodes should be smooth. The electrodes of the present inventionare preferably coated on at least the surface from which the electricfield emanates with a continuous crystalline metal nitride, as describedin more detail above.

The electrodes can be hollow, to allow cooling by liquid or gas, or theelectrodes can be solid, to allow for thermoelectric or any other typeof conductive cooling. Cooling could also be accomplished by cooling theelectroporation chamber itself, apart from cooling the electrodes.

Preferably, the flow electroporation chamber is disposable. A detaileddescription of three embodiments of the electroporation chamber of thepresent invention is provided below.

In one embodiment, the flow electroporation chamber is constructed ofclear polyvinyl chloride, and contains two opposing electrodes spaced adistance of approximately 7 mm apart. The electroporation chamber is amodification of a chamber obtained from BTX Electronic Company of SanDiego, Calif. However, when this electroporation chamber is usedcontinuously, it overheats and the survival rate of the cells processedby the apparatus decreases over time. To correct the overheating problemthat occurred when the apparatus was used in a continuous flow manner, acontinuous flow electroporation chamber was designed. A detaileddescription of the structure of the continuous flow electroporationchamber is provided below.

FIGS. 3 through 8 show one embodiment of the flow electroporationchamber 72 of the present invention. As can be seen in FIG. 3, the flowelectroporation chamber 72 includes a housing 100 having two electrodes102 inset on opposing sides of the housing 100 of the electroporationchamber 72. The housing 100 includes an inlet channel 104 at one end andan outlet channel 106 at the other. The inlet 104 and outlet 106channels include connectors 108 and 109 respectively, preferably of themale Luer variety. The connectors 108 and 109 are hollow and form theinlet 104 and outlet 106 channels into the interior of theelectroporation chamber 72.

As seen in FIGS. 4 and 5, an internal chamber 110 extends most of thelength of the housing 100 and is sized to receive the two electrodes102. The internal chamber 110 includes beveled surfaces 111 forreceiving the internal edges of the electrodes 102. The internal chamber110 is thus formed by the internal surfaces of the electrodes 102 andthe internal surfaces of the housing 100. The internal chamber 110 isconnected to the inlet 104 and outlet 106 channels.

As can be seen in FIGS. 7 and 8, the electrodes 102 of theelectroporation chamber 72 of FIGS. 3 to 6 are comprised of flat,elongated, hollow shells. The electrodes of the present invention arepreferably coated on at least the surface from which the electric fieldemanates with a continuous crystalline metal nitride, as described inmore detail above. The electrodes 102 include cooling inlets 112 andcooling outlets 114 at their ends. As described above, the rear surfacesof the electrodes 102, or the surface to the left in FIG. 7, fits flushagainst the beveled surface 111 of the housing 100.

The electroporation chamber 72 is designed such that the cell suspensionto be subjected to electroporation enters the electroporation chamber 72through the inlet 104 and expands to fill the internal chamber 110. Asthe red blood cell suspension flows through the internal chamber 110 apulse or charge is administered across the width of the internal chamber110.

To maintain a relatively constant temperature during the electroporationprocess, cooling fluid or cooling gas is pumped in the cooling inlet 112and out the cooling outlet 114 so that the electrodes 102 are maintainedat approximately 4° C.

FIGS. 9 and 10 display a second embodiment of the flow electroporationchamber 172. As can be seen in FIGS. 9 and 10, the flow electroporationchamber 172 includes a hollow housing 200 substantially rectangular inshape. Two electrodes 202 are inserted into the interior of the housing200 directly opposite one another, flush against the housing 200 walls.The flow electroporation chamber 172 further comprises an inlet channel204 at one end and an outlet channel 206 at the other end of the housing200. The inlet 204 and outlet 206 channels include connectors 208 and209 which are attached by tubing 216 to a cell suspension supply thatsupplies the cell suspension, i.e. the IHP-red blood cell suspension, tothe electroporation chamber 172. The connectors 208 and 209 and inlet204 and outlet 206 channels serve to direct the cell suspension into andout of the housing 200.

As can be seen in FIG. 10, one end of the inlet channel 204 and one endof the outlet channel 206 extends into the interior of the housing 200forming an internal chamber 210. The internal chamber 210 is thus formedby the internal surfaces of the electrodes 202, the internal surfaces ofthe housing 200 and the internal surfaces of the of the inlet 204 andoutlet 206 channels.

As can be seen in FIGS. 9 and 10, the electrodes 202 of the flowelectroporation chamber 172 comprise flat, elongated, hollow shells. Theelectrodes 202 include cooling inlets 212 and cooling outlets 214 attheir ends, through which a gas or fluid may be pumped through theelectrodes 202 to maintain a constant temperature duringelectroporation. The electrodes 202 are connected to a pulse generatorby cables 220. The electrodes of the present invention are preferablycoated on at least the surface from which the electric field emanateswith a continuous crystalline metal nitride, as described in more detailabove.

As with the chamber described above, the electroporation chamber 172 ofFIGS. 9 and 10 is designed such that the suspension to be subjected toelectroporation enters the electroporation chamber 172 through the fluidinlet 204 and expands to fill the internal chamber 210. As the red bloodcells suspension flows through the internal chamber 210, a pulse orcharge is administered across the width of the internal chamber 210between the electrodes 202. To maintain a relatively constanttemperature during the electroporation process, cooling fluid or coolinggas is pumped in the cooling inlet 212 and out the cooling outlet 214 ofthe electrodes 202 through the connectors 208 and 209 so that theelectrodes 202 are maintained at approximately 4° C. It is also possiblethat the inlet channel 204, outlet channel 206 and connectors 208 and209 can be made as a solidly integrated glass part, rather than separatecomponents.

It is contemplated that the flow electroporation chamber 172 maybeconstructed from drawn glass or any other highly polished material. Itis preferable that the interior surface of the electroporation chamber172 be as smooth as possible to reduce the generation of surfaceturbulence. Drawn glass components are highly consistent with perfectsurface finishes. Furthermore, they are stable and inert to bloodcomponents. They are also relatively inexpensive, which is desirable fora disposable electroporation chamber.

The electrodes may also be comprised of drawn glass, electroplated withcolloidal gold. Again, the surfaces of the electrodes should be highlyfinished, highly conductive, yet biologically inert. Gold electroplateis durable and inexpensive. Fluidic connection can be accomplished usingcommonly available parts. The electrodes of the present invention arepreferably coated on at least the surface from which the electric fieldemanates with a continuous crystalline metal nitride, as described inmore detail above.

The flow electroporation chamber may be constructed either as a part ofthe entire flow encapsulation apparatus, or as an individual apparatus.The flow electroporation apparatus may then be connected to acommercially available plasmaphoresis machine for encapsulation ofparticular cell populations. For example, the flow electroporationchamber may be connected to commercially available plasmaphoresisequipment by electronic or translational hardware or software.Optionally, a pinch-valve array and controller driven by a PC programcan also be used to control the flow electroporation apparatus.Similarly, current power supplies are capable of establishing the powerlevels needed to run the flow electroporation chamber or flowencapsulation apparatus.

A third embodiment of a continuous flow electroporation chamber will nowbe described with reference to FIGS. 14-20. Referring first to FIGS.14-16, a support member 300 is comprised of flexible silicone rubber.The support structure 300 is essentially diamond shaped and comprises anupper end 301 and a lower end 302. A major portion of the supportstructure 300 has a grid-like “waffle” pattern formed on it, comprisedof thicker rib sections 303 and thinner sections 304 intermediate theribs 303. Along the marginal edges of the support structure 300, aplurality of tabs 305 are provided, each having a hole 306 formedtherethrough.

A channel 308 extends between the upper end 301 and the lower end 302 ofthe support structure and lies along the major axis of the supportstructure 300. The channel 308 comprises opposed channel walls 310, 312connected by a base 314. At the upper end 301 of the support structure300 the channel 308 opens into a circular cavity 318. A hole 320 isformed in the center of the circular cavity 318. An outlet aperture 322is provided at the upper end of the circular cavity 318. In a likemanner, the lower end of the channel 308 opens into a circular cavity324 formed in the lower end 302 of the support structure 300. A hole 326is formed through the support structure in the center of the cavity 324,and an inlet aperture 328 is provided at the lower end of the circularcavity 324.

A pair of continuous band electrodes 330A, 330B comprised of conductivemetallic tape or foil are located on the support structure 300. Each ofthe electrodes 330A, 330B has a portion which is disposed within thechannel 308 and which runs substantially the entire length of thechannel 308. As can perhaps best be seen in FIG. 16, electrodes 330A,330B are received in opposing recesses 332 formed in the side walls 310,312 of the channel 308. Adjacent the upper and lower ends of the channel308, each of the continuous band electrodes 330A, 330B exits the channel308 through a close fitting slit formed in the channel walls. Thecontinuous band electrodes 330A, 330B then curve outward and extendsubstantially parallel to the periphery of the support member 300 andspaced inward therefrom. Along the midline of the support structure 300and adjacent its outer edges, a slack portion 334 is provided in each ofthe continuous band electrodes 330A, 330B, for the purposes to bedescribed below.

On either side of the channel 308 and immediately adjacent thereto, aplurality of generally rectangular holes 340 are formed. As will be morefully explained below, the holes 340 are located to optionallyaccommodate Peltier thermo-electric elements for cooling purposes. Oneither side of the channel 308 adjacent its upper and lower ends,circular holes 342 are provided which, as will be shown, are adapted toreceive capstans for tensioning the continuous band electrodes 330A,330B. Along the midline of the support structure 300 and adjacent itsouter edges, a pair of holes 344 which, as will be more fully explainedbelow, are adapted to receive electrical contacts therethrough forcharging the electrodes 330A, 330B. The electrodes of the presentinvention are preferably coated on at least the surface from which theelectric field emanates with a continuous crystalline metal nitride, asdescribed in more detail above.

Referring now to FIG. 17, the support member 300 is mounted to atransparent polycarbonate frame 350. The frame 350 comprises a planarfront wall 352. Interior side walls 354 extend rearward from the lateraledges of the planar front wall 352. A rearward opening channel 356 isformed between the two interior side walls 354. At the rear edges of theinterior side walls 354, a pair of back walls 358 extend outward. A pairof exterior side walls 360 extend forward from the outer edges of theback walls 358. Forward opening channels 362 are formed between theexterior side walls 360 and the interior side walls 354. Rods 363removably mounted in each of the forward opening channels 362 provide aconvenient means for hanging fluid storage bags within the channels.

The support structure 300 is mounted to the back surface of the frontwall 352 of the polycarbonate frame 350. The support structure 300 isadhesively bonded to the frame 350 such that.the front wall 352 of theframe 350 seals the open upper end of the channel 308 formed in the faceof the support structure 300. Thus enclosed, the channel 308 defines afluid passage or “flow cell” 364. In addition, the support structure 300and associated.portion of the frame 350 define an electroporationchamber 366.

Referring further to FIG. 17, a support column 370 has a generallyrectangular cross section. In the front face 371 of the support column370 a cavity 372 is formed which conforms to the shape and depth of thesupport structure 300. Spaced on either side and along the major axis ofthe cavity 372, a plurality of bismuth telluride Peltier thermo-electricelements 374 are fixedly mounted in the cavity and project forward fromthe base of the cavity 372. The Peltier thermo-electric elements 374 arein thermal communication with a heat sink 375 mounted inside the supportcolumn 370. An electric fan 376 mounted in an adjacent portion of thesupport column 370 creates a flow of air through the column to dissipateheat away from the heat sink 375.

Adjacent the upper and lower ends of the cavity 372 and spaced to eitherside of the center line are capstans 377. Adjacent the outer edges ofthe cavity 372 and located along the minor axis of the cavity are a pairof electrode contacts 378. Located just inside the perimeter of thecavity 372 are eight locator pins 379, two of the locator pins 379 beingsituated along each of the four walls of the diamond-shaped cavity. Atthe upper and lower ends of the cavity 372 and located on the major axisof the cavity are a pair of hollow, porous, polymeric cylinders 380. Thecylinders 380 are preferably formed of inert foamed polyethylene (suchas Porex) with a pore size permitting passage of gas but not liquid. Aswill be more fully explained hereinbelow, these gas-permeable, liquidimpermeable cylinders function as a means for removing bubbles fromfluid passing thereover.

The dimensions of the polycarbonate frame 350 are such that the supportcolumn 370 is snugly received within the rearward opening channel 356 ofthe frame. As the frame 350 is positioned onto the support column 370,the support structure 300 mounted to the back surface of the front wall352 of the frame 350 fits within the cavity 372 formed in the front face371 of the support column 370. A shelf 381 is located on the front face371 of the support column 370 immediately below the cavity 372 tosupport the lower edge of the polycarbonate frame 350.

With the frame 350 thus mounted to the support column 370, the variouselements associated with the cavity 372 and the support column 370cooperatively engage the support structure 300 as shown in FIG. 19.Specifically, the thermo-electric cooling elements 374 project throughthe holes 340 in the support structure 300 and contact the walls of thechannel 308. The capstans 377 extend through the holes 342 in the upperand lower ends of the support structure 300. The electrode contacts 378project through the holes 344 in the support structure 300. The locatorpins 379 are received within the corresponding holes 306 in the tabs 305of the support member 300. And the gas-permeable, liquid impermeablecylinders 380 extend through the holes 320, 326 in the cavities 318, 324at the upper and lower ends 301, 302 of the support structure 300.

Referring now to FIG. 20, at least one of the capstans 377 supportingeach of the electrodes 330A, 330B is tensioned such as by tensioningmeans 382 to maintain the continuous band electrodes in a taut state. Ascan also be seen in FIG. 20, each electrode contact 378 has a slot 383formed in its face, and the slack section 334 of the associatedcontinuous band electrode 330A or 330B is threaded through this slot. Amotor 384 in driving engagement with each electrode contact 378 can beoperated to rotate the electrode contact, thereby winding the electrode330A or 330B around a portion of the contact and taking up the slack.This winding action serves the additional function of increasing surfacecontact between the electrode contact 378 and its associated electrode330A or 330B, thereby enhancing the electrical connection to theelectrodes.

The gas permeable, liquid impermeable cylinders 380 at the upper andlower ends of the flow cell 364 (only the upper of which is shown inFIG. 20) are in fluid communication with a vacuum source by way of acoupling 388 and tubing 390. Also shown in FIG. 20, the heat sinks 375dissipate the heat collected by the thermo-electric cooling elements374.

Control of fluid flow along the appropriate flow paths into and out ofthe flow cell 364 is accomplished by peristaltic pumping means 392 andsolenoid-activated pinch valves 394, mounted in the support column 350.The pumping means 392 and pinch valves 394 operate under control ofappropriate algorithms in computer means (not shown) operably connectedthereto.

A cooled plate 396 is mounted on the side of the support column 370. Acooling bag 398 retained in the channel 362 of the frame 350 is held inintimate contact with this plate 396 to cool treated fluids followingelectroporation. Depending upon the circumstances and the biologicalsubstance being treated, the plate 396 may optionally be heated tomaintain the contents of the bag 398 at a predetermined temperatureabove that of the ambient.

FIG. 21 illustrates a self-contained electroporation apparatus 400. Theapparatus 400 comprises a cart 402 which serves as a housing and supportstructure. The support column 370 with electroporation chamber 366 ismounted to the cart and extends upward therefrom. The cart 402 has achassis structure 404, which is provided with wheels 406 to facilitatetransport of the cart 402 from one location to another. Mounted to thechassis structure 404 are power supply capacitors 408. A power supplyheatsink 410 is in thermal communication with the power supplycapacitors 408 to dissipate the heat generated by the power supplycapacitors.

A circuit board computation means 412 is also mounted within the chassisstructure 404. The circuit board computation means 412 is powered by apower supply circuit board 414 mounted within the chassis structure 404adjacent the circuit board computation means. A power supply heatsink416 in thermal communication with the power supply circuit board 414dissipates the heat generated by the power supply circuit board. Acooling fan 418 mounted at the lower end of the front panel 420 of thechassis structure 404 pulls air through the chassis structure to drawheat away from the heatsinks 410, 416.

A system status display 422 operatively associated with the circuitboard computation means 412 is mounted to the front panel 420 of thecart 402. Control switches 424 for setting various parameters of thecircuit board computation means 412 are mounted to the front panel 420of the cart 402 below the system status display 422.

Mounted within the top panel 428 of the cart 402 is a centrifuge bowl430. A centrifuge drive motor 432 mounted within the chassis structure404 is in driving engagement with the centrifuge bowl 430. Thecentrifuge bowl 430 includes a rotary connector 434 through which bloodis input into the centrifuge bowl.

Treatment of biological particles in the self-contained electroporationapparatus 400 comprising the electroporation chamber 366 of the thirdembodiment will now be described with reference to FIG. 21. A bloodsupply bag 450 is hung on a rod 363 within one of the channels 362 ofthe frame 350. A tubing 452 transports the blood to the centrifuge bowl430, where it is introduced into the centrifuge bowl through a rotaryconnector 434. The blood is centrifuged to separate the red blood cellsfrom the plasma, white blood cells, and waste. The red blood cells arethen admixed with the substance to be encapsulated. The admixture istransported via a tubing 454 and introduced into the inlet aperture 328at the lower end of the cell 364. The admixture is caused to flow upwardthrough the flow cell 364 between the electrodes 330A, 330B. Theelectrodes are charged in a pulsed manner, as hereinabove described withrespect to the second embodiment. Gases in the admixture resulting fromelectrolysis are removed by the gas-permeable, liquid impermeablecylinders 380 at the upper and lower ends of the cell. The treatedadmixture exits the outlet aperture 324 at the upper end of the cell,and an outlet tubing 456 transports the treated admixture to a coolingbag 460 suspended on a rod 363 within another one of the channels 362 ofthe frame 350 and in contact with the cooled plate 396. The fluid isthen conveyed to a post-treatment cooling and storage bag 462 suspendedon the rod 363 next to the cooling bag 460.

Pump speeds (and hence flow rates), valve operation, centrifugeoperation, operation of the Peltier thermo-electric elements, and pulsedcharging of the electrodes are all controlled by the circuit boardcomputation means 412. Ideally, the processing rate of the centrifugebowl 430 is matched to the flow rate of the flow cell 364. However, toaccommodate any mismatch, a reservoir may optionally be provided betweenthe centrifuge bowl 430 and the flow cell 364. Thus if the centrifugebowl 430 processes the blood faster than the flow cell 364 can processit, the reservoir will hold any excess admixture until the flow cell can“catch up.” Similarly, if the centrifuge bowl 430 processes the bloodslower than the flow cell 364 can process it, the circuit boardcomputation means 412 can initially accumulate admixture in thereservoir. Then when the centrifuge bowl 430 has processed a sufficientvolume of blood, admixture can be transported from the reservoir to theflow cell 364. By the time the volume of admixture in the reservoir hasbeen depleted, the centrifuge bowl will have completed processing thedesired quantity of blood.

An optional feature of the electroporation apparatus 400 hereinabovedescribed is that the series of Peltier thermo-electric cooling elements374 can be individually controllable, such that cooling elements 374 atone location along the flow cell 364 can provide a greater or lesserdegree of cooling than other cooling elements 374 at other locationsalong the flow cell 364. Since the biological particles are being heatedas they move along the flow cell 364, more cooling may be necessarycloser to the discharge end of the flow cell 364 than is necessaryadjacent the input end. Providing individual control over the variousthermoelectric cooling elements 374 permits accommodation of thesevariations. The various thermoelectric cooling elements 374 can becontrolled either by placing thermal sensors at various locations alongthe flow cell, inputting the sensed temperatures into the circuit boardcomputation means 412, and controlling the various thermo-electriccooling elements in response to the sensed temperatures. Or, the variousthermo-electric cooling elements 374 can be controlled according to apredetermined “average” temperature variance of the biological particlesalong the flow cell. Other methods for controlling variousthermo-electric cooling elements 374 will occur to those skilled in theart.

As will be appreciated by those familiar with the art, there are severalreasons why it is not desirable to re-use an electroporation cell.First, the possibility exists that infectious components could betransferred to other patients. Further, electrical performance of theelectrode surfaces would degrade due to the high voltage potentialsacross these surfaces, thereby increasing the potential for arcing. Toprevent these and other problems, a feature of this third disclosedembodiment provides a means for ensuring that the cell is not re-used.At the termination of the procedure, and before the frame 350 is removedfrom the support column 370, the motors 384 in driving engagement withthe electrode contacts 378 are automatically actuated to over-rotate,tensioning the electrodes 330A, 330B beyond their tensile strength andbreaking them. With the electrodes 330A, 330B thus broken, re-use of thecell is impossible.

A known risk associated with electroporation apparatus is theunintentional production of gases by electrolysis. Overpressuresresulting from the unwanted buildup of such gases have been known toresult in explosive expression. To minimize this possibility, thepresent invention employs a flow cell 364 defined on three sides by softsilicone rubber. In the event of transient overpressures, the elasticityof the support structure 300 will accommodate expansion of the flow cell364 and thereby reduce the possibility of explosion. In addition, theflow cell 364 is sandwiched tightly between the support column 370 andthe polycarbonate frame 350, providing further protection against anypossible explosive expression.

While this invention has been described in specific detail withreference to the disclosed embodiments, it will be understood that manyvariations and modifications may be effected within the spirit and scopeof the invention as described in the appended claims.

Cell Washing Apparatus

FIG. 24 is a cutaway schematic view of a cell washing apparatus 500utilizing filtration dialysis, preferably, counter current filtrationdialysis. The intact apparatus has a top and sides which completelycontain the internal elements of the apparatus. Another aspect of thepresent invention is a cell washing apparatus that utilizescounter-current dialysis through a porous membrane to remove the IHPsolution and substitute therefore a solution that is compatible with redblood cells including, but not limited to, normal saline. As shown inFIG. 24, the cell washing apparatus 500 comprises a first reservoir 505which contains cells that have been electroporated. In the case of cellsthat have been electroporated in the presence of IHP, these cells willhave been passed through the electroporation chamber 72 and will be in asolution containing excess IHP. The electroporated cells are then pumpedthrough tubing 515 by pump 510 in the direction of the arrow. The cellsuspension are introduced into the cell washing apparatus 500 at tubingentrance 520 which is located in housing cell washing apparatus housing523. The cell path within the apparatus 500 is defined by a cell plate526 which has an ridges 525 which define a labyrinth through which thecell suspension will travel.

A preferred labyrinth is shown in FIG. 25 which shows a side view of thecell plate 526 showing the ridges on the plate that define thelabyrinth. The cell plate 526 is forced against the first side 577 of asemi-permeable membrane 575 at a force great enough so that the cellsare forced along the labyrinth defined by the ridges 525. It is to beunderstood that the labyrinth defined by the ridges 525 can be any shapeso long as the cell suspension is in contact with the semi-permeablemembrane 575. The cell suspension is therefore in intimate contact withthe semi-permeable membrane 575 while it is passing through the cellwashing apparatus 500.

The semi-permeable membrane 575 has pores that are large enough to allowthe solution and any dissolved constituents of the solution to passthrough the membrane but will not allow the cells in the solution topass through the membrane. The semipermeable membrane can be anymaterial that is compatible with the cells that are in the cellsuspension. Semipermeable membranes that can be used in the cell washingapparatus of the present invention include, but are not limited to,polypropylene (Travenol Laboratories) cellulose diacetate (AsahiMedical), polyvinyl alcohol (Kuraray, polymethylmethacrylate (Toray),and polyvinyl chloride (Cobe Laboratories). For red blood cells, thepores in the semipermeable membrane should be no larger than 1 micron indiameter but may be much smaller in diameter. The cells travel along thelabyrinth defined by ridges 525 until the cell suspension exits theapparatus 500 at the exit tube 530. With regard to a cell suspensionwith IHP therein, the cell suspension is then pumped back to reservoir505 and is recirculated through the apparatus 500 until the level of IHPin the bathing solution has dropped to an acceptable level.

On the other side of semi-permeable membrane 575, is an identical salineplate 536 which has identical ridges 555 to those ridges on cell plate526. The saline plate is pushed against the second side 578 of thesemi-permeable membrane 575 thereby defining a labyrinth that is themirror image of the labyrinth defined by ridges 525. A wash solutionthat is biocompatible with the cells, for example, saline, is pumpedfrom the reservoir 540 containing the biocompatible fluid by pump 565through tube 567 to cell washing apparatus 500 at wash solution entrance550.

It is to be understood that the wash solution can be any solution thatis biocompatible with the cells that are being washed. This includes,but is not limited to, isotonic saline, hypertonic saline, hypotonicsaline, Krebs-Ringer bicarbonate buffer, Earle's balanced salts, Hanks'balanced salts, BES, BES-Tris, HEPES, MOPS, TES, and Tricine. Cellculture media can be used as a wash solution, including, but not limitedto, medium 199, Dulbecco's modified eagle's medium, CMRL-1066, minimumessential medium (MEM), and RPMI-1640. In addition, the resealingsolutions as defined herein can be used as a wash solution. Finally, anycombination of the aforementioned solutions can be used as a washsolution.

The biocompatible solution is pumped through the apparatus by pump 565following the labyrinth defined by the ridges 555 until thebiocompatible solution exits the cell washing apparatus 500 at exit 560.The biocompatible solution is then discarded through drain 570. It isimportant to note that the apparatus 500 will be most efficient if thebiocompatible solution is pumped in an opposite direction to that of thesolution containing the cells. However, it is contemplated in thisinvention that biocompatible solution can be pumped in the samedirection as the solution containing the cells.

Using the IHP containing cell suspension from electroporated cells as anexample, as both solutions are pumped through cell washing apparatus500, the cell suspension solution containing the IHP will diffusethrough the semi-permeable membrane 575 and, simultaneously, thebiocompatible solution, will diffuse in the opposite direction throughthe semi-permeable membrane 575. As this diffusion continues, the cellsuspension solution will gradually be diluted and replaced with thebiocompatible solution until the level of IHP is at an acceptable level.

The cell washing apparatus 500 can optionally have a thermal electricelement 580 attached to the outside of the cell plate 526 and theoutside of wash solution plate 536. It is to be understood that thethermal electric element 580 can be attached to either one or both ofthe outside of plates 526 and 536. The thermal electric element 580 canbe used to cool the solutions or can be used to warm the solutionsduring the wash cycle. Thus, it is to be understood that if the cellwashing apparatus is used with the thermal electric elements attachedthereto, the incubator 78 is not required because the cells will beresealed when warmed in the cell washing apparatus which will serve asan incubator. The biocompatible wash solution can be the resealingbuffer. It is to be understood that temperature can be controlled byother methods such as a water bath.

The shape of the cell washing apparatus 500 can be any shape including around container wherein the inner portion of the round containercontains the cell suspension and is separated from the outer portion ofthe round container by the semipermeable membrane 575. The roundcontainer 500 could be rotated slowly to help force the solutioncontaining the cells through the semipermeable membrane 575 therebyremoving the contaminating material.

The cell washing apparatus can be comprised of any material that isbiocompatible with the cells that are to be washed in the apparatus. Thecell plate 526 and the wash plate 536 can be manufactured from flexiblesilicone rubber.

Another embodiment of a cell washing apparatus that can be used tosubstitute for the centrifuge for washing the electroporated cells isshown in FIG. 26. In this second embodiment of the cell washingapparatus 600, the central feature of the cell washing apparatus is anelastomeric cell 605 which is made from elastomeric material such assilicone rubber. Turning now to FIG. 27, the elastomeric cell 605 is amolded piece with a semi-permeable membrane 610 in the center of theelastomeric cell 605. On either side of the semi-permeable membrane 610are horizontal indentations 615 which form a labyrinth and run theentire length of the elastomeric cell.

As shown in FIG. 26, the elastomeric cell 605 has inlet port 625 forintroducing a wash solution and an outlet port 630 for removing the washsolution and an inlet port 635 for introducing the cells with theelectroporation fluid and an outlet port 640 for removing the cells withthe electroporation fluid. Thus, the wash solution is introduced on oneside of the semipermeable membrane 610 in the elastomeric cell 605, iscirculated through the labyrinth and exits at outlet port 640. Theelectroporation solution containing the electroporated cells isintroduced on the other side of the semipermeable membrane 610, iscirculated through the labyrinth and exits at outlet port 640.

It is to be understood that the semi-permeable membrane 610 completelyseparates the two sides and that any communication between the two sidesis through the semi-permeable membrane 610. The semi-permeable membranehas pores that allow the solutions to pass through the membrane 610, butdoes not allow particles, such as cells to pass through thesemipermeable membrane 610. The semipermeable membrane can be anymaterial that is compatible with the cells that are in the cellsuspension. Semipermeable membranes that can be used in the cell washingapparatus of the present invention include, but are not limited to,polypropylene (Travenol Laboratories) cellulose diacetate (AsahiMedical), polyvinyl alcohol (Kuraray, polymethylmethacrylate (Toray),and polyvinyl chloride (Cobe Laboratories). For red blood cells, thepores in the semipermeable membrane should be no larger than 1 micron indiameter but may be much smaller in diameter.

The elastomeric cell can be placed into a frame 655 and side 660 can berotated on hinges 665 and 666 so that the side 660 holds the elastomericcell 605 against side 665 thereby wedging the elastomeric cell tightlybetween side 660 and side 665. Side 660 is a thermal electric elementwhich is capable of heating or cooling the elastomeric cell 610. Side665 is a pulsatile mechanism with a roller 670 which travels on belt 675and can sequentially squeeze the elastomeric cell as the roller travelsaround the belt 675 and sequentially puts pressure on flexible rods 677which run vertically the height of side 665.

In operation, the elastomeric cell is placed into the frame 655 and theside 660 (the thermal electric element) is closed onto the elastomericcell 605. Of course, the side 660 can be a plate without the thermalelectric element. On the first side 615, the inlet is attached to thewash solution tube which is attached to a wash solution reservoir (notshown). Outlet 630 is connected to a drain tube (not shown). On theother side of the elastomeric cell, inlet 635 is connected to thereservoir containing the cells and electroporation fluid (not shown).Outlet 640 is connected to a tube which returns the cells andelectroporation fluid to the cell reservoir.

In operation, the peristaltic activator 670 gently pumps on the washsolution side thereby forcing the fluids from the inlet side to theoutlet side. Optionally, the two solutions can be pumped through the twolabyrinths by external pumps in a manner similar to that shown in cellwashing apparatus 500. Because the parastaltic activator is pressing onthe elastomeric cell, the transfer of fluid across the semipermeablemembrane 650 is enhanced by mass transfer action. This action iscontinued until the electroporation fluid is essentially replaced by thewash fluid.

Application of IHP Treated Red Blood Cells

The present invention provides a novel method for increasing theoxygen-carrying capacity of erythrocytes. In accordance with the methodof the present invention, the IHP combines with hemoglobin in a stableway, and shifts its oxygen releasing capacity. Erythrocytes withIHP-hemoglobin can release more oxygen per molecule than hemoglobinalone, and thus more oxygen is available to diffuse into tissues foreach unit of blood that circulates. Under ordinary circumstances, IHP istoxic and cannot be tolerated as an ordinary drug. Attachment of IHP tohemoglobin in this novel procedure, however, neutralizes its toxicity.In the absence of severe chronic blood loss, treatment with acomposition prepared in accordance with the present method could resultin beneficial effects that persist for approximately ninety days.

Another advantage of IHP-treated red blood cells is that they do notlose the Bohr effect when stored. Normal red blood cells that have beenstored by conventional means do not regain their maximum oxygen carryingcapacity for approximately 24 hours. This is because the DGP in normalred blood cells diffuses away from the hemoglobin molecule duringstorage and must be replaced by the body after transfusion. In contrast,red blood cells treated according to the present invention are retaintheir maximum oxygen carrying capacity during storage and therefore candeliver maximum oxygen to the tissues immediately after transfusion intoa human or animal.

The uses of IHP-treated RBC's is quite extensive including the treatmentof numerous acute and chronic conditions including, but not limited tohospitalized patients, cardiovascular operations, chronic anemia, anemiafollowing major surgery, coronary infarction and associated problems.chronic pulmonary disease, cardiovascular patients, autologoustransfusions, as an enhancement to packed red blood cells transfusion(hemorrhage, traumatic injury, or surgery). congestive heart failure,myocardial infarction (heart attack), stroke, peripheral vasculardisease, intermittent claudication, circulatory shock, hemorrhagicshock, anemia and chronic hypoxia, respiratory alkalemia, metabolicalkalosis, sickle cell anemia, reduced lung capacity caused bypneumonia, surgery, pneumonia, trauma, chest puncture, gangrene,anaerobic infections, blood vessel diseases such as diabetes, substituteor complement to treatment with hyperbaric pressure chambers,intra-operative red cell salvage, cardiac inadequacy, anoxia—secondaryto chronic indication, organ transplant, carbon monoxide, nitric oxide,and cyanide poisoning.

Treating a human or animal for any one or more of the above diseasestates is done by transfusing into the human or animal betweenapproximately 0.5 and 6 units (1 unit=500 ml) of IHP-treated blood thathas been prepared according to the present invention. In certain cases,there may be a substantially complete replacement of all the normalblood in a patient with IHP-treated blood. The volume of IHP-treated redblood cells that is administered to the human or animal will depend uponthe indication being treated. In addition, the volume of IHP-treated redblood cells will also depend upon concentration of IHP-treated red bloodcells in the red blood cell suspension. It is to be understood that thequantity of IHP red blood cells that is administered to the patient isnot critical and can vary widely and still be effective.

IHP-treated packed RBC's are similar to normal red blood cells in exceptthat the IHP-treated packed red blood cells can deliver 2 to 3 times asmuch oxygen to tissue per unit. A physician would therefore chose toadminister a single unit of IHP-treated packed red blood cells ratherthan 2 units of the normal red blood cells. IHP-treated packed red bloodcells could be prepared in blood processing centers analogously to thepresent blood processing methods, except for the inclusion of aprocessing step where the IHP is encapsulated in the cells.

While this invention has been described in specific detail withreference to the disclosed embodiments, it will be understood that manyvariations and modifications may be effected within the spirit and scopeof the invention as described in the appended claims.

What is claimed is:
 1. A flow electroporation chamber for electricalstimulation of particles in a saline solution, comprising a housinghaving an inlet, an outlet, and internal walls defining a particleelectrical stimulation chamber; the chamber being configured to receivea continuous flow of particles from the inlet; electrodes disposed alongthe walls of the chamber, the electrodes in electrical communicationwith a source of electrical energy, whereby flowing particles in thechamber are subjected to an electric field; and the electrodes eachfurther comprising an external surface wherein at least a portion of theexternal surface of one of the electrodes has a continuous crystallinemetal nitride coating.
 2. The flow electroporation chamber of claim 1,wherein the electrodes disposed along the walls of the chamber compriseelectrodes exposed into the chamber.
 3. The flow electroporation chamberof claim 1, wherein the source of electrical energy is adapted to supplypulsed electrical energy.
 4. The flow electroporation chamber of claim1, wherein the electrical energy is pulsed.
 5. The flow electroporationchamber of claim 1, wherein the electrical energy is a variable flux. 6.The flow electroporation chamber of claim 1, wherein at least a portionthe surface of the electrodes corresponding to the electrical field hasa crystalline metal nitride coating.
 7. The flow electroporation chamberof claim 1, wherein the continuous crystalline metal nitride coating istitanium nitride, titanium aluminum nitride, chromium nitride orzirconium nitride.
 8. The flow electroporation chamber of claim 7,wherein the continuous crystalline metal coating is titanium nitride.